17 DNA Repair Gene MMutations aand · analysis of mutations. Finally, we take a look at DNA repair...

The Genetic Legacy of ChernobylEarly on the morning of April 26, 1986, unit 4 of the Cher-nobyl nuclear power plant in northern Ukraine exploded,creating the worst nuclear disaster in history. The explo-sion blew off the 2000-ton metal plate that sealed the topof the reactor and ignited hundreds of tons of graphite,which burned uncontrollably for 10 days. The exactamount of radiation released in the explosion and ensuingfire is still unknown, but a minimum estimate is 100 mil-

lion curies, equal to a medium-sized nuclear strike. Aplume of radioactive particles blew west and north fromthe crippled reactor, raining dangerous levels of radiationdown on thousands of square kilometers. Regions as faraway as Germany and Norway were affected; even Japanand the United States received measurable increases inradiation.

Immediately after the accident, 31 people, mostly fire-fighters who heroically battled the blaze, died of acute radi-ation sickness. More than 400,000 workers later toiled to

472

• The Genetic Legacy of Chernobyl

• The Nature of MutationThe Importance of Mutations

Categories of Mutations

Types of Gene Mutations

Mutation Rates

• Causes of MutationsSpontaneous Replication Errors

Spontaneous Chemical Changes

Chemically Induced Mutations

Radiation

• The Study of MutationsThe Analysis of Reverse Mutations

Detecting Mutations with theAmes Test

Radiation Exposure in Humans

• DNA RepairMismatch Repair

Direct Repair

Base-Excision Repair

Nucleotide-Excision Repair

Other Types of DNA Repair

Genetic Diseases and FaultyDNA Repair

GGeennee MMuuttaattiioonnss aannddDDNNAA RReeppaaiirr17

This is photo legend x 26 picas width for opening chapter photofor Chapter 17. This is legend copy area for Chapter opening photo forChapter Seventeen allowing 4lines, if more space is needed crop photoat top to allow for deeper legend here. (Volodymyr Repik/AP).

Gene Mutations and DNA Repair 473

bury radioactive and chemical wastes from the accident andto entomb the remains of the disabled reactor in a steel andconcrete sarcophagus. Many of these workers are now ill,suffering from a variety of problems including immunesuppression, increased rates of cancer, and reproductivedisorders.

Radiation is a known mutagen, causing damage to DNA.More than 13,000 children in the area surrounding Cher-nobyl were exposed to the radioactive isotope iodine-131;many had exposures 400 times the maximum annual radi-ation exposure recommended for workers in the nuclearindustry. The rate of thyroid cancer among children in theUkraine is now 10 times the pre-Chernobyl levels. Chromo-some mutations have been detected in the cells of manypeople who resided near Chernobyl at the time of the acci-dent, and birth defects in the population have increasedsignificantly.

To examine germ-line mutations (those passed on tofuture generations) resulting from the Chernobyl accident,geneticists collected blood samples from 79 families whoresided in heavily contaminated districts. These familiesincluded children born in 1994 who had not been exposedto radiation but who might possess mutations acquiredfrom their parents. DNA sequences from these parents andchildren were analyzed, allowing the researchers to identifypossible germ-line mutations. The germ-line mutation ratein these families was found to be twice as high as that in acontrol group of families in Britain. Furthermore, themutation rate was correlated with the level of surface radia-tion: families in which the parents had resided in more-contaminated districts had higher mutation rates than thosefrom less-contaminated districts.

This chapter is about the infidelity of DNA — abouthow errors arise in genetic instructions and how thoseerrors are sometimes repaired. The Chernobyl catastropheillustrates one cause of mutations (radiation) and the detri-mental effects that DNA damage can have.

We begin with a brief examination of the differenttypes of mutations, including their phenotypic effects, howthey may be suppressed, and mutation rates. The next sec-tion explores how mutations spontaneously arise in thecourse of replication and afterward, as well as how chemi-cals and radiation induce mutations. We then consider theanalysis of mutations. Finally, we take a look at DNA repairand some of the diseases that arise when DNA repair isdefective. Throughout the chapter, it will be useful to keepin mind that mutations, by definition, are inherited changesin the DNA sequence — they must be passed on. Mutationrequires both that the structure of a DNA molecule bechanged and that this change is replicated.

More information about thehealth effects of radiation released in the Chernobylaccident

The Nature of MutationDNA is a highly stable molecule that replicates with amaz-ing accuracy (see Chapters 10 and 12), but changes in DNAstructure and errors of replication do occur. A mutation isdefined as an inherited change in genetic information; thedescendants may be cells produced by cell division or indi-vidual organisms produced by reproduction.

The Importance of MutationsMutations are both the sustainer of life and the cause ofgreat suffering. On the one hand, mutation is the source ofall genetic variation, the raw material of evolution. Withoutmutations and the variation that they generate, organismscould not adapt to changing environments and would riskextinction. On the other hand, most mutations have detri-mental effects, and mutation is the source of many humandiseases and disorders.

Much of genetics focuses on how variants produced bymutation are inherited; genetic crosses are meaningless if allindividuals are identically homozygous for the same alleles.Mutations serve as important tools of genetic analysis; the so-lution to almost any genetic problem begins with a good set ofmutants. Much of Gregor Mendel’s success in unraveling theprinciples of inheritance can be traced to his use of carefullyselected variants of the garden pea; similarly, Thomas HuntMorgan and his students discovered many basic principles ofgenetics by analyzing mutant fruit flies ( FIGURE 17.1).

Mutations are also useful for probing fundamental bio-logical processes. Finding mutations that affect different com-ponents of a biological system and studying their effects canoften lead to an understanding of the system. This method, re-ferred to as genetic dissection, is analogous to figuring outhow an automobile works by breaking different parts of a carand observing the effects—for example, smash the radiatorand the engine overheats, revealing that the radiator cools theengine. The disruption of function in individual organismsbearing particular mutations likewise can be a source of in-sight into biological processes. For example, geneticists havebegun to unravel the molecular details of development bystudying mutations that interrupt various embryonic stages inDrosophila (see Chapter 21). Although this method of break-ing “parts” to determine their function might seem like acrude approach to understanding a system, it is actually verypowerful and has been used extensively in biochemistry, de-velopmental biology, physiology, and behavioral science (butthis method is not recommended for learning how your carworks).

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www.whfreeman.com/pierce

ConceptsMutations are heritable changes in the geneticcoding instructions of DNA. They are essential tothe study of genetics and are useful in many otherbiological fields.

474 Chapter 17474 Chapter 17

Categories of MutationsIn multicellular organisms, we can distinguish between twobroad categories of mutations: somatic mutations and germ-line mutations. Somatic mutations arise in somatic tissues,which do not produce gametes ( FIGURE 17.2). These muta-tions are passed on to other cells through the process of mi-tosis, which leads to a population of genetically identical cells(a clone). The earlier in development that a somatic mutationoccurs, the larger the clone of cells within that individual or-ganism that will contain the mutation.

Because of the huge number of cells present in a typicaleukaryotic organism, somatic mutations must be numer-ous. For example, there are about 1014 cells in the human

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body. If a mutation arises only once in every million celldivisions (a fairly typical rate of mutation), hundreds ofmillions of somatic mutations must arise in each person.The effect of these mutations depends on many factors,including the type of cell in which they occur and the devel-opmental stage at which they arise. Many somatic muta-tions have no obvious effect on the phenotype of theorganism, because the function of the mutant cell (even thecell itself) is replaced by that of normal cells. However, cellswith a somatic mutation that stimulates cell division canincrease in number and spread; this type of mutation cangive rise to cells with a selective advantage and is the basisfor all cancers (see Chapter 21).

17.1 Morgan and his students discovered many principles ofheredity by studying mutation in Drosophila melanogaster.Shown here are several common mutations.

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17.2 There are two basic classes ofmutations: somatic mutations and germ-line mutations.◗

Wild type

Bar eyes

Vestigial wings Curly wings Bithorax Dichaete

White eyesMiniature (wings) Curved Kidney

Mitosis

Sexualreproduction

Somaticmutation

Germ-linemutation

Somatictissue

Mutant cell

Populationof mutant cells

All cellscarry mutation

No cellscarry mutation

Germ-linetissue

1 Somatic mutations occurin nonreproductive cells…

3 Germ-line mutations occur incells that give rise to gametes.

4 Meiosis and sexual reproductionallow germ-line mutations to bepassed to approximately half themembers of the next generation,…

5 …who will carry themutation in all their cells.

2 …and are passed to other cellsthrough mitosis, creating a cloneof cells having the mutant gene.


Germ-line mutations arise in cells that ultimately pro-duce gametes. These mutations can be passed to futuregenerations, producing individual organisms that carry themutation in all their somatic and germ-line cells (see Figure17.2). When we speak of mutations in multicellular orga-nisms, we’re usually talking about germ-line mutations. Insingle-cell organisms, however, there is no distinction betweengerm-line and somatic mutations, because cell division resultsin new individuals.

Historically, mutations have been partitioned intothose that affect a single gene, called gene mutations, andthose that affect the number or structure of chromosomes,called chromosome mutations. This distinction arose becausechromosome mutations could be observed directly, bylooking at chromosomes with a microscope, whereas genemutations could be detected only by observing their phe-notypic effects. Now, with the development of DNAsequencing, gene mutations and chromosome mutationsare distinguished somewhat arbitrarily on the basis of thesize of the DNA lesion. Nevertheless, it is useful to use theterm chromosome mutation for a large-scale genetic alter-ation that affects chromosome structure or the number ofchromosomes and the term gene mutation for a relativelysmall DNA lesion that affects a single gene. This chapterfocuses on gene mutations; chromosome mutations werediscussed in Chapter 9.

Types of Gene MutationsThere are a number of ways to classify gene mutations.Some classification schemes are based on the nature of thephenotypic effect — whether the mutation alters the aminoacid sequence of the protein and, if so, how. Other schemes

are based on the causative agent of the mutation, and stillothers focus on the molecular nature of the defect. Themost appropriate scheme depends on the reason for study-ing the mutation. Here, we will categorize mutations pri-marily on the basis of their molecular nature, but we willalso encounter some terms that relate the causes and thephenotypic effects of mutations.

Base substitutions The simplest type of gene mutationis a base substitution, the alternation of a single nucleotidein the DNA ( FIGURE 17.3a). Because of the complemen-tary nature of the two DNA strands (see Figure 10.14),when the base of one nucleotide is altered, the base of thecorresponding nucleotide on the opposite strand also willbe altered in the next round of replication. A base substitu-tion therefore usually leads to a base-pair substitution.

Base substitutions are of two types. In a transi-tion, a purine is replaced by a different purine or, alterna-tively, a pyrimidine is replaced by a different pyrimidine ( FIGURE 17.4). In a transversion, a purine is replaced by apyrimidine or a pyrimidine is replaced by a purine. The num-ber of possible transversions (see Figure 17.4) is twice thenumber of possible transitions, but transitions usually arisemore frequently.

Insertions and deletions The second major class of genemutations contains insertions and deletions — the addi-tion or the removal, respectively, of one or more nucleotidepairs ( FIGURE 17.3b and c). Although base substitutionsare often assumed to be the most common type of muta-tion, molecular analysis has revealed that insertions anddeletions are more frequent. Insertions and deletions within

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AGT GTA GAT AGT GCA GAT AGT AGA TCGGTT

T

AGT GAG ATC

T

GGG GGG GGG GGGCGT CGT T GTC

(a) Base substitution (b) Insertion (c) Deletion

One codon changed

Original DNA sequence

A base substitutionalters a single codon.

An insertion or a deletion alters the readingframe and may change many codons.

Transitions Possible base changes

Transversions

Purine

A GG A

T CC T

A CA TG CG T

C AC GT AT G

Purine

Pyrimidine PurinePyrimidinePyrimidine

Purine Pyrimidine

17.3 Three basic types of gene mutations are base substitutions,insertions, and deletions.◗

17.4 A transition is the substitution of apurine for a purine or apyrimidine for a pyrimidine; a transversionis the substitution of apyrimidine for a purine or a purine for a pyrimidine.

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476 Chapter 17

sequences that encode proteins may lead to frameshift mu-tations, changes in the reading frame (see p. 000 in Chapter15) of the gene. The initiation codon in mRNA sets thereading frame: after the initiation codon, other codons areread as successive nonoverlapping groups of three nu-cleotides. The addition or deletion of a nucleotide usuallychanges the reading frame, altering all amino acids encodedby codons following the mutation (see Figure 17.3b and c).Many amino acids can be affected; so frameshift mutationsgenerally have drastic effects on the phenotype. Not all in-sertions and deletions lead to frameshifts, however; becausecodons consist of three nucleotides, insertions and deletionsconsisting of any multiple of three nucleotides will leave thereading frame intact, although the addition or removal ofone or more amino acids may still affect the phenotype.These mutations are called in-frame insertions and dele-tions, respectively.

ConceptsGene mutations consist of changes in a singlegene and may be base substitutions (a single pairof nucleotides is altered) or insertions or deletions(nucleotides are added or removed). A basesubstitution may be a transition (substitution oflike bases) or a transversion (substitution of unlikebases). Insertions and deletions often lead to achange in the reading frame of a gene.

Expanding trinucleotide repeats In 1991, an entirelynovel type of mutation was discovered. This mutationoccurs in a gene called FMR-1 and causes fragile-X syn-drome, the most common hereditary cause of mental retar-dation. The disorder is so named because, in specially treatedcells of persons having the condition, the tip of the X chro-mosome is attached only by a slender thread ( FIGURE 17.5).The FMR-1 gene contains a number of adjacent copies of thetrinucleotide CGG. The normal FMR-1 allele (not contain-ing the mutation) has 60 or fewer copies of this trinucleotidebut, in persons with fragile-X syndrome, the allele may har-

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17.5 The fragile-X chromosome is associatedwith a characteristic constriction (fragile site) onthe long arm. (Visuals Unlimited.)

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Examples of genetic diseases caused by expandingtrinucleotide repeats

Table 17.1

Number of Copies of Repeat

Repeated Normal Disease Disease Sequence Range Range

Spinal and bulbar muscular atrophy CAG 11–33 40–62

Fragile-X syndrome CGG 6–54 50–1500

Jacobsen syndrome CGG 11 100–1000

Spinocerebellar ataxia (several types) CAG 4–44 21–130

Autosomal dominant cerebellar ataxia CAG 7–19 37–�220

Myotonic dystrophy CTG 5–37 44–3000

Huntington disease CAG 9–37 37–121

Friedreich ataxia GAA 6–29 200–900

Dentatorubral-pallidoluysian atrophy CAG 7–25 49–75

Myoclonus epilepsy of the CCCGCCCGCG 2–3 12–13Unverricht-Lundborg type*

*Technically not a trinucleotide repeat but does entail a multiple of three nucleotides that expands andcontracts in similar fashion to trinucleotide repeats.


bor hundreds or even thousands of copies. Mutations inwhich copies of a trinucleotide may increase greatly in num-ber are called expanding trinucleotide repeats.

Expanding trinucleotide repeats have been found inseveral other human diseases (Table 17.1). The number ofcopies of the trinucleotide repeat often correlates with theseverity or age of onset of the disease. The number of copiesof the repeat also correlates with the instability of trinu-cleotide repeats — when more repeats are present, the prob-ability of expansion to even more repeats increases. Thisinstability leads to a phenomenon known as anticipation(see p. 000 in Chapter 5), in which diseases caused bytrinucleotide-repeat expansions become more severe ineach generation. Less commonly, the number of trinu-cleotide repeats may decrease within a family.

How an increase in the number of trinucleotides pro-duces disease symptoms is not yet clear. In several of thediseases (e.g., Huntington disease), the trinucleotide CAGexpands within the coding part of a gene, producing a toxicprotein that has extra glutamine residues (the amino acidencoded by CAG). In other diseases (e.g., fragile-X syn-drome and myotonic dystrophy), the repeat is outside thecoding region of the gene and therefore must have someother mode of action. At least one disease (a rare type ofepilepsy) has now been associated with an expanding repeatof a 12-bp sequence. Although this repeat is not a trinu-cleotide, it is included as a type of expanding trinucleotidebecause its repeat is a multiple of three.

The mechanism that leads to the expansion of trinu-cleotide repeats is still unclear. Strand slippage in DNA repli-cation (see Figure 17.14) and crossing over betweenmisaligned repeats (see Figure 17.15) are two possible sourcesof expansion. Single-stranded regions of some trinucleotiderepeats are known to fold into hairpins ( FIGURE 17.6) andother special DNA structures. Such structures may promotestrand slippage in replication and may prevent these errorsfrom being recognized and corrected, as described later inthis chapter in the section on mismatch repair.

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(a)

(b)

(c)

(d)

(e)

(f)

(g)

Mis-pairedbases

1 2 3 4 5 6 7 8

1 2 3 4 5 6 7 8

1 2 3 4 5 6 7 8

9 10 11 12 13

9 10 11 12 13

1 2 3 4 5 6 7 8 9 10 11 12 13

9 10 11 12 13

5

1 2 3 4 5 6 7 8

1 23

8

4 6

7

C GA

GTC GTC GTC GTC GTC GTC GTC GTCCAG CAG CAG CAG CAG CAG CAG CAG

GTC GTC GTC GTC GTC GTC GTC GTC GTC GTC GTC GTC GTCCAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG

GTC GTC GTC GTC GTC GTC GTC GTCCAG CAG CAG CAG CAG CAG CAG

GTC GTC GTC GTC GTC GTC GTC GTC

GTC GTC GTC GTC GTC GTC GTC GTCCAG CAG CAG CAG CAG CAG CAG CAG

GTC GTC GTC GTC GTC GTC GTC GTCCAG CAG CAG

GTC GTC GTCCAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG

C GA

C

GC

GAA

AA

C GA

C

GC

GAA

AA

G

CG

C

G

CG

C

1 This DNA moleculehas eight copies ofa CAG repeat.

2 The two strandsseparate…

3 …and replicate.

4 During replication, a hairpin formson the newly synthesized strand,…

5 …causing part of the templatestrand to be replicated twice andincreasing the number of repeatson the newly synthesized strand.

6 The two strands of the new DNAmolecule separate,…

7 …and the strand with extra CAGcopies serves as a templatefor replication.

8 The resulting DNA molecule contains five additional copiesof the CAG repeat.

ConceptsExpanding trinucleotide repeats are regions ofDNA that consist of repeated copies of threenucleotides. Increased numbers of trinucleotiderepeats are associated with several geneticdiseases.

Phenotypic effects of mutations Mutations havea variety of phenotypic effects. The effect of a mutationmust be considered with reference to a phenotype againstwhich the mutant can be compared, which is usually thewild-type phenotype — that is, the most common pheno-type in natural populations of the organism. For example,most Drosophila melanogaster in nature have red eyes; so

17.6 The number of copies of a trinucleotidemay increase by strand slippage in replication.◗

478 Chapter 17

red eyes are considered the wild-type eye color; any othergenetically determined eye color in fruit flies is consideredto be a mutant. A mutation that alters the wild-type pheno-type is called a forward mutation, where as a reverse muta-tion (a reversion) changes a mutant phenotype back intothe wild type.

Geneticists use special terms to describe the phenotypiceffects of mutations. A base substitution that alters a codonin the mRNA, resulting in a different amino acid in the pro-tein, is referred to as a missense mutation ( FIGURE 17.7a).A nonsense mutation changes a sense codon (one thatspecifies an amino acid) into a nonsense codon (one thatterminates translation; FIGURE 17.7b). If a nonsense mu-tation occurs early in the mRNA sequence, the protein willbe greatly shortened and will usually be nonfunctional.A silent mutation alters a codon but, thanks to the redun-dancy of the genetic code, the codon still specifies the sameamino acid ( FIGURE 17.7c). A neutral mutation is a mis-sense mutation that alters the amino acid sequence of theprotein but does not change its function. Neutral mutationsoccur when one amino acid is replaced by another that ischemically similar or when the affected amino acid has littleinfluence on protein function.

Loss-of-function mutations cause the complete orpartial absence of normal function. A loss-of-functionmutation so alters the structure of the protein that the pro-tein no longer works correctly or the mutation can occur inregulatory regions that affect the transcription, translation,or splicing of the protein. Loss-of-function mutations arefrequently recessive, and diploid individuals must behomozygous for the mutation before they can exhibit theeffects of the loss of the functional protein. In contrast, again-of-function mutation produces an entirely new traitor it causes a trait to appear in inappropriate tissues or atinappropriate times in development. These mutations arefrequently dominant in their expression. Still other types of

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mutations are conditional mutations, which are expressedonly under certain conditions, and lethal mutations, whichcause premature death.

Suppressor mutations A suppressor mutation is agenetic change that hides or suppresses the effect of anothermutation. This type of mutation is distinct from a reversemutation, in which the mutated site changes back into theoriginal wild-type sequence ( FIGURE 17.8). A suppressormutation occurs at a site that is distinct from the site of theoriginal mutation; thus, an individual organism with a sup-pressor mutation is a double mutant, possessing both theoriginal mutation and the suppressor mutation but exhibit-ing the phenotype of an unmutated wild type.

Geneticists distinguish between two classes of suppres-sor mutations: intragenic and intergenic. An intragenicsuppressor is in the same gene as that containing the muta-tion being suppressed and may work in several ways. Thesuppressor may change a second nucleotide in the samecodon that was altered by the original mutation, producinga codon that specifies the same amino acid as the original,unmutated codon ( FIGURE 17.9). Intragenic suppressorsmay also work by suppressing a frameshift mutation. If theoriginal mutation is a one-base deletion, then the additionof a single base elsewhere in the gene will restore the formerreading frame (see Figure 17.9). Consider the followingnucleotide sequence in DNA and the amino acids that itencodes:

DNA AAA TCA CTT GGC GTA CAAAmino acids Phe Ser Glu Pro His Val

Suppose a one-base deletion occurs in the first nucleotideof the second codon. This deletion shifts the reading frameby one nucleotide and alters all the amino acids that followthe mutation.

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TCAAGT

TCAAGT

UCA

Ser

UUA

Leu

UAA UCG

Ser

TAAATT

TTAAAT

TCGAGC

Stop codon

DNA

DNA

mRNA

Protein

No mutation (a) Missense mutation (b) Nonsense mutation (c) Silent mutation

Wild-type proteinproduced.

The new codon encodes adifferent amino acid; there is achange in amino acid sequence.

The new codon is a stopcodon; there is prematuretermination of translation.

The new codon encodes thesame amino acid; there is nochange in amino acid sequence.

17.7 Base substitutions can cause (a) missense, (b) nonsense,and (c) silent mutations.◗


One-nucleotide deletion

AAA TXCAC TTG GCG TAC AAPhe Val Asn Arg Stop

If a single nucleotide is added to the third codon (the sup-pressor mutation), the reading frame is restored, althoughtwo of the amino acids differ from those specified by theoriginal sequence.

One-nucleotide duplication

AAA CAC TTTX GGC GTA CAAPhe Val Lys Pro His Val

Similarly, a mutation due to an insertion may be suppressedby a subsequent deletion in the same gene.

A third way in which an intragenic suppressor maywork is by making compensatory changes in the protein.A first missense mutation may alter the folding of a poly-peptide chain by changing the way in which amino acidsin the protein interact with one another. A second miss-ense mutation at a different site (the suppressor) may recre-ate the original folding pattern by restoring interactionsbetween the amino acids.

Intergenic suppressors, in contrast, occur in a genethat is different from the one bearing the original mutation.These suppressors sometimes work by changing the waythat the mRNA is translated. In the example illustrated in( FIGURE 17.10), the original DNA sequence is AAC (UUGin the mRNA) and specifies leucine. This sequence mutatesto ATC (UAG in mRNA), a termination codon. The ATCnonsense mutation could be suppressed by a mutation ina gene that encodes a tRNA molecule by changing theanticodon on the tRNA so that it is capable of pairingwith the UAG termination codon. For example, the genethat encodes the tRNA for tyrosine (tRNATyr), which has theanticodon AUA, might be mutated to have the anticodonAUC, which will then pair with the UAG stop codon.Instead of translation terminating at the UAG codon, tyro-sine would be inserted into the protein and a full-lengthprotein would be produced, although tyrosine wouldnow substitute for leucine. The effect of this change woulddepend on the role of this amino acid in the overall struc-ture of the protein, but the effect is likely to be less detri-mental than the effect of the nonsense mutation, whichwould halt translation prematurely.

Because cells in many organisms have multiple copiesof tRNA genes, other unmutated copies of tRNATyr would

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MutationsA– B–

Wild typeA+ B+

MutationA–

Suppressormutation B–

Forwardmutation A–

Reverse ofmutation A–

Genotype:

Red eyes White eyes Red eyes

2 A reverse mutationrestores the wild-typegene and the phenotype.

3 A suppressor mutation occursat a site different from thatof the original mutation…

4 … and produces anindividual that possessesboth the originalmutation and thesuppressor mutation…

5 …but has thewild-type phenotype.

1 A forward mutationchanges the wild typeinto a mutant phenotype.

17.8 Relation of forward, reverse, and suppressor mutations.◗

17.9 An intragenic suppressor mutation occurs in the samegene that contains the mutation being suppressed.◗

AAT

UUA

Leu

UUU

Phe

CUU

Leu

AAA GAAMutationIntragenicsupressormutation

DNA

mRNA

Protein

2 A second mutationat a different site inthe same gene…

3 …may restore the original amino acid.

1 A missense mutationalters a single codon.

480 Chapter 17

remain available to recognize the tyrosine codons. How-ever, we might expect that the tRNAs that have undergonea suppressor mutation would also suppress the normal ter-mination codons at the ends of coding sequences, resultingin the production of longer-than-normal proteins, but thisevent does not usually take place. Mutations in tRNA genescan also suppress missense and frameshift mutations.

Intergenic suppressors can also work through genicinteractions (see p. 000 in Chapter 5). Polypeptide chains

which are produced by two genes may interact to pro-duce a functional protein. A mutation in one gene may alter the encoded polypeptide so that the interaction isdestroyed and then a functional protein is no longer pro-duced. A suppressor mutation in the second gene mayproduce a compensatory change in its polypeptide there-fore restoring the original interaction. Characteristics ofsome of the different types of mutations are summarizedin Table 17.2.

AAC

Leu

AUC

AUA

Tyr

Transcription

TranscriptionTranslation

AAC

UUG

UUG

TTG

AUC

Tyr

UAG

Base-substitutionmutation

Termination oftranslation

Translation

AAC

UAG

UAG

TTG

ATCTAG

Stop codon

Transcription

Second base-substitution mutation

Translation

ATC

UAG

TAG

ATCTAG

TATATA

TAGATC

(a)

mRNA

DNA Site 1(first mutation) Site 2

Full-length,functional

protein

Full-length,functional

protein

Ribosome

(b)

Shortened,nonfunctional

protein

(c)

tRNA

tRNA

1 With the wild-type sequence,…

2 Leu is incorporatedinto a protein.

3 A base substitutionat one site producesa prematurestop codon,…

4 …which haltsprotein synthesis,resulting in a non-functional protein.

5 At site 2 is a geneencoding tyrosine-tRNA.

6 Normal transcriptionproduces a tRNA with ananticodon AUA (whichwould pair with the tyrosinecodon UAU in translation).

7 If a base substitutionintroduces anincorrect base (G),…

8 …the resulting mutanttRNA has anticodonAUC (instead of AUA),…

9 …which can pair withthe stop codon UAG.

10 Translation continuespast the stop codon,Tyr is incorporated into the protein.

17.10 An intergenic suppressor mutation occurs in a differentgene from the one bearing the original mutation. (a) The wild-typesequence produces a full-length, functional protein. (b) A base substitutionat a site in one gene produces a premature stop codon, resulting in ashortened, nonfunctional protein. (c) A base substitution at a site in another gene, which in this case encodes tRNA, alters the anticodon of tRNATyr

so that tRNATyr can pair with the stop codon produced by the originalmutation, allowing tyrosine to be incorporated into the protein andtranslation to continue. Tyrosine replaces the leucine residue present inthe original protein.

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Descriptions and illustrations ofdifferent types of mutations

Mutation RatesThe frequency with which a gene changes from the wildtype to a mutant is referred to as the mutation rate and isgenerally expressed as the number of mutations per bio-logical unit, which may be mutations per cell division, per

Characteristics of different types of mutationsTable 17.2

Type of Mutation Definition

Base substitution Changes the base of a single DNA nucleotide

Transition Base substitution in which a purine replaces a purine or apyrimidine replaces a pyrimidine

Transversion Base substitution in which a purine replaces a pyrimidine or apyrimidine replaces a purine

Insertion Addition of one or more nucleotides

Deletion Deletion of one or more nucleotides

Frameshift mutation Insertion or deletion that alters the reading frame of a gene

In-frame deletion Insertion or deletion of a multiple of three nucleotides or insertion that does not alter the reading frame

Expanding trinucleotide Repeated sequence of three nucleotides (trinucleotide) repeats in which the number of copies of the trinucleotide

increases

Forward mutation Changes the wild-type phenotype to a mutant phenotype

Reverse mutation Changes a mutant phenotype back to the wild-type phenotype

Missense mutation Changes a sense codon into a different sense codon, resultingin the incorporation of a different amino acid in the protein

Nonsense mutation Changes a sense codon into a nonsense codon, causingpremature termination of translation

Silent mutation Changes a sense codon into a synonymous codon, leavingunchanged the amino acid sequence of the protein

Neutral mutation Changes the amino acid sequence of a protein without alteringits ability to function

Loss-of-function mutation Causes a complete or partial loss of function

Gain-of-function mutation Causes the appearance of a new trait or function or causes theappearance of a trait in inappropriate tissues or atinappropriate times

Lethal mutation Causes premature death

Suppressor mutation Suppresses the effect of an earlier mutation at a different site

Intragenic suppressor Suppresses the effect of an earlier mutation within the mutation same gene

Intergenic suppressor Suppresses the effect of an earlier mutation in mutation another gene

ConceptsA suppressor mutation overrides the effect of anearlier mutation at a different site. An intragenicsuppressor mutation occurs within the same gene,as that containing the original mutation, whereasan intergenic suppressor mutation occurs in adifferent gene.


482 Chapter 17

Achondroplasia is an inheritedautosomal dominant condition thatcauses diminished growth in the longbones of the legs, leading to dwarfism.Several years ago, the gene forachondroplasia was identifiedand cloned. If two people withachondroplasia marry, and eachof them is heterozygous forachondroplasia (has one of twopossible copies of the gene), chancesare that two of every four children thatthey have will also be heterozygousand dwarfs. On average, one child inevery four born to the couple will notinherit the achondroplasia gene andwill be of average height, and onechild in four will be homozygous forthe gene. Homozygosity for this geneis lethal, and these children usually diein infancy.

A researcher who helped identifythe gene understandably felt that hehad made a significant contributionby allowing short-statured parents theoption of aborting fetuses with thelethal double dose of the gene. Tohis surprise, shortly after news of thediscovery was published, he receiveda call from one member of anachondroplasia couple, askingwhether it was possible to test forboth the presence and the absenceof the gene. The couple wanted thisinformation, they said, because theyplanned to abort not just all fetuseshomozygous for the achondroplasiagene, but any completely unaffectedones as well. They were intent onhaving only short-statured children likethemselves.

This case poses at least two majorconflicts for genetic professionals.First, there is the conflict betweenrespect for parental autonomy, whichwould ordinarily encourage accedingto the parents’ request for assistanceand information, and the medicalprofessional’s desire not to visitharm on a child. Children born withachondroplasia frequently must

undergo a series of surgical proceduresto correct serious bone problems.Throughout life, they also face manysocial and physical obstacles becauseof their short stature. Is it right forparents to deliberately bring a childinto existence with this condition? Is itappropriate for health professionals toassist such efforts? How do we balancerespect for parental autonomy againstnonmaleficence?

Matters become more complexwhen we realize that some peoplewith achondroplasia reject the ideathat any harm is being done by theparents in this case. They maintainthat most of the problems that theyface are socially constructed and aredue to society’s marginalization andneglect of those who are different.Some also reject medical or genetic“solutions” to their problems. Theproper response, they believe, is notto prevent the birth of a child with agenetic condition but to eliminate thesocial handicaps and discriminatoryattitudes. Thus, the parents in thiscase may be driven not merely bytheir personal wishes but by acommitment to social justice.

Traditional, nondirective geneticcounseling has assumed that peopleseek prenatal testing to prevent the

The New GeneticsETHICS • SCIENCE • TECHNOLOGY

birth of a child with a genetic disease,to prepare for the birth and treatmentof a child with a recognized geneticdisorder, or to reconsider theirreproductive plans. What this casereveals is that genetics is opening upthe possibility of shaping our children’slives in ways that go far beyond whatis normally associated with the healingrole. Somewhat less dramatic, butperhaps more worrisome is the factthat the identification of the geneticbasis of many traits that are notconsidered diseases (e.g., height,intelligence, temperament) will offerparents a new range of choices in the“genetic design” of their children. Atthis moment, research is underway toidentify and replace disease-causinggenes in human embryos. In thefuture, such embryonic gene therapywill open up the possibility ofenhancing children’s capabilities.Beginning with genes that improvea child’s resistance to cancer or AIDS,genetic interventions may make itpossible to increase a child’s height,stamina, or IQ. Science could offerparents who yearn for a championbasketball player or world-classswimmer the means to realize theirdreams.

As complex as it may seem, thescience here is the easy part. Far moredifficult are the ethical questions. Tobegin with, there is the question ofwhether we will ever have enoughknowledge to “play God” in thisway. Do we dare alter the course ofhuman evolution? The history oftwentieth-century science is litteredwith well-intended technologies—fromDDT to nuclear power—that eventuallybrought unforeseen harms. Will ourgenetic interventions follow this path?Will our clumsy attempts to “improve”the human genome unleash anepidemic of new genetic diseases?And what of the child’s rights in allthis? Is it fair to “engineer” a child intoa parent’s dream of perfection?

Achondroplasia

A family of three who have achondroplasia.(Gail Burton/AP.)

by Ron Green


gamete, or per round of replication. For example, the mu-tation rate for achondroplasia (a type of hereditarydwarfism) is about four mutations per 100,000 gametes,usually expressed more simply as 4 � 10�5. In contrast,mutation frequency is defined as the incidence of a spe-cific type of mutation within a group of individual organ-isms. For achondroplasia, the mutation frequency in theUnited States is about 2 � 10�4, which means that about 1of every 20,000 persons in the U.S. population carries thismutation.

Mutation rates are affected by three factors. First, theydepend on the frequency with which primary changes takeplace in DNA. Primary change may arise from sponta-neous molecular changes in DNA or it may be induced bychemical or physical agents in the environment.

A second factor influencing the mutation rate is theprobability that, when a change takes place, it will berepaired. Most cells possess a number of mechanisms torepair altered DNA; so most alterations are corrected be-fore they are replicated. If these repair systems are effec-tive, mutation rates will be low; if they are faulty, mutationrates will be elevated. Some mutations increase the overallrate of mutation at other genes; these mutations usuallyoccur in genes that encode components of the replicationmachinery or DNA repair enzymes.

A third factor, one that influences our ability to calcu-late mutation rates, is the probability that a mutation willbe recognized and recorded. When DNA is sequenced, all

mutations are potentially detectable. In practice, however,sequencing is expensive; so mutations are usually detectedby their phenotypic effects. Some mutations may appearto arise at a higher rate simply because they are easier todetect.

Mutation rates vary among organisms and amonggenes within organisms (Table 17.3), but we can drawseveral general conclusions about mutation rates. First,spontaneous mutation rates are low for all organismsstudied. Typical mutation rates for viral and bacterialgenes range from about 1 to 100 mutations per 10 billioncells (1 � 10�8 to 1 � 10�10). The mutation rates formost eukaryotic genes are a bit higher, from about 1 to 10mutations per million gametes (1 � 10�5 to 1 � 10�6).These higher values in eukaryotes may be due to the factthat the rates are calculated per gamete, and several celldivisions are required to produce a gamete, whereas mu-tation rates in prokaryotic cells and viruses are calculatedper cell division.

Within each major class of organisms, mutation ratesvary considerably. These differences may be due to differ-ing abilities to repair mutations, unequal exposures to mu-tagens, or biological differences in rates of spontaneouslyarising mutations. Even within a single species, sponta-neous rates of mutation vary among genes. The reason forthis variation is not entirely understood, but some regionsof DNA are known to be more susceptible to mutationthan others.

Mutation rates of different genes in different organismsTable 17.3

Organism Mutation Rate Unit

Bacteriophage T2 Lysis inhibition 1 � 10�8 Per replicationHost range 3 � 10�9

Escherichia coli Lactose fermentation 2 � 10�7 Per cell divisionHistidine requirement 2 � 10�8

Neurospora crassa Inositol requirement 8 � 10�8 Per asexual sporeAdenine requirement 4 � 10�8

Corn Kernel color 2.2 � 10�6 Per gamete

Drosophila Eye color 4 � 10�5 Per gameteAllozymes 5.14 � 10�6

Mouse Albino coat color 4.5 � 10�5 Per gameteDilution coat color 3 � 10�5

Human Huntington disease 1 � 10�6 Per gameteAchondroplasia 1 � 10�5

Neurofibromatosis 1 � 10�4

(Michigan)Hemophilia A (Finland) 3.2 � 10�5

Duchenne muscular 9.2 � 10�5

dystrophy (Wisconsin)

484 Chapter 17

Causes of MutationsMutations result from both internal and external factors.Those that are a result of natural changes in DNA structureare termed spontaneous mutations, whereas those thatresult from changes caused by environmental chemicals orradiation are induced mutations.

Spontaneous Replication ErrorsReplication is amazingly accurate: fewer than one in abillion errors are made in the course of DNA synthesis(Chapter 12). However, spontaneous replication errors dooccasionally occur.

The primary cause of spontaneous replication errorswas formerly thought to be tautomeric shifts, in which thepositions of protons in the DNA bases change. Purine andpyrimidine bases exist in different chemical forms calledtautomers ( FIGURE 17.11a). The two tautomeric forms ofeach base are in dynamic equilibrium, although one form ismore common than the other. The standard Watson andCrick base pairings — adenine with thymine, and cytosinewith guanine — are between the common forms of thebases, but, if the bases are in their rare tautomeric forms,other base pairings are possible ( FIGURE 17.11b).

Watson and Crick proposed that tautomeric shiftsmight produce mutations, and for many years their pro-posal was the accepted model for spontaneous replicationerrors, but there has never been convincing evidence thatthe rare tautomers are the cause of spontaneous mutations.Furthermore, research now shows little evidence of thesestructures in DNA.

Mispairing can also occur through wobble, in whichnormal, protonated, and other forms of the bases are

◗

◗

ConceptsMutation rate is the frequency with which aspecific mutation arises, whereas mutationfrequency is the incidence of a mutation within adefined group of individual organisms. Rates ofmutations are generally low and are affected byenvironmental and genetic factors.

H3C

NH2

O

O

T

H3C

O

O

T

H

O

C

H

OH

N

GN

NH

H

H

NH2

GN N

NN

AN

AN N

N

N

HH

N

N

N

H

N

H

N

H

N

H

H HN

H

N

N

O

C

N

H

N

N

N

N N

HN

H

H

TN

N

H3C O

O

TN

N

H3C O

O

CN

N

O

H

H

N

CN

O

H

H

N

G

N

H

O

N

H

H

N

N

A

N

H

N

H

H

N

H

A

N

H

N

H

H

H

N

H

H

G

NO

N

H

H

N

N

H

N

H

H

O

H

H

H

H

H

H

H

H

H H

H

H

H

H

H

H H

N

N

H

H

H

H

N

N

N

N

(a)

(b)

Proton shiftCommon forms

Standard base-pairing arrangements

Anomalous base-pairing arrangements

Thymine

Thymine (common form) Adenine (common form)

Cytosine (common form)

Cytosine (rare form)

Thymine (common form)

Guanine (common form)

Guanine

Cytosine

Adenine

Rare forms

Adenine (commom form)

Guanine (rare form)

17.11 Purine and pyrimidine bases exist indifferent forms called tautomers. (a) A tautomericshift occurs when a proton changes its position, resultingin a rare tautomeric form. (b) Standard and anomalousbase-pairing arrangements occur if bases are in the raretautomeric forms. Base mispairings due to tautomericshifts were originally thought to be a major source oferrors in replication, but such structures have not beendetected in DNA, and most evidence now suggeststhat other types of anomalous pairings (see Figure 17.14)are responsible for replication errors.

◗


able to pair because of flexibility in the DNA helical struc-ture ( FIGURE 17.12). These structures have been detectedin DNA molecules and are now thought to be responsiblefor many of the mispairings in replication.

When a mismatched base has been incorporated into anewly synthesized nucleotide chain, an incorporated erroris said to have occurred. Suppose that, in replication,thymine (which normally pairs with adenine) mispairs withguanine through wobble ( FIGURE 17.13). In the nextround of replication, the two mismatched bases separate,and each serves as template for the synthesis of a newnucleotide strand. This time, thymine pairs with adenine,producing another copy of the original DNA sequence. Onthe other strand, however, the incorrectly incorporated gua-nine serves as the template and pairs with cytosine, produc-ing a new DNA molecule that has an incorporated error — a

◗

◗

C�G pair in place of the original T�A pair (a T�A:C�Gbase substitution). The original incorporated error leads toa replication error, which creates a permanent mutation,because all the base pairings are correct and there is nomechanism for repair systems to detect the error.

Mutations due to small insertions and deletions also mayarise spontaneously in replication and crossing over. Strandslippage may occur when one nucleotide strand forms asmall loop ( FIGURE 17.14). If the looped-out nucleotides areon the newly synthesized strand, an insertion results. At thenext round of replication, the insertion will be incorporatedinto both strands of the DNA molecule. If the looped-outnucleotides are on the template strand, then there is a dele-tion on the newly replicated strand, and this deletion will beperpetuated in subsequent rounds of replication.

During normal crossing over, the homologoussequences of the two DNA molecules align, and crossingover produces no net change in the number of nucleotidesin either molecule. Misaligned pairing may cause unequalcrossing over, which results in one DNA molecule with aninsertion and the other with a deletion ( FIGURE 17.15).Some DNA sequences are more likely than others toundergo strand slippage or unequal crossing over. Stretchesof repeated sequences, such as trinucleotide repeats orhomopolymeric repeats (more than five repeats of the samebase in a row), are prone to strand slippage. Stretches withmore repeats are more likely to undergo strand slippage.Duplicated or repetitive sequences may misalign duringpairing, leading to unequal crossing over. Both strand slip-page and unequal crossing over produce duplicated copiesof sequences, which in turn promote further strand slippageand unequal crossing over. This chain of events may explainthe phenomenon of anticipation often observed forexpanding trinucleotide repeats.

◗

◗

G

NTN

N

H3C O

O

O

C N

O

N

H

H

N

A

N

H

N

H

H

H

N

H

H

N

NN

H

H

N

N

H

H

N

Thymine–guanine wobble

Non-Watson-Crick base pairing

Cytosine–adenine protonated wobble

17.12 Nonstandard base pairings can occur asa result of the flexibility in DNA structure. Thymineand guanine can pair through wobble between normalbases. Cytosine and adenine can pair through wobblewhen adenine is protonated (has an extra hydrogen).

◗

17.13 Wobble base pairing leads to a replicated error.◗

TTCG

TTCG

TTCG

AAG C

AAG C TTGCAAC G

TTCGAAG C

TCCGAGG C

TTC GA G GC

AGGCDNA Wild type

Wild type

Wild type

Mutant

3 At the next round of replication, theguanine nucleotide pairs with cytosine,leading to a transition mutation.

1 DNA strands separatefor replication.

2 Thymine on the original template strand base pairs withguanine through wobble, leading to an incorporated error.

ConceptsSpontaneous replication errors arise from alteredbase structures and from wobble base pairing.Small insertions and deletions may occur throughstrand slippage in replication and through unequalcrossing over.

486 Chapter 17

Spontaneous Chemical ChangesIn addition to spontaneous mutations that arise in replica-tion, mutations also result from spontaneous chemicalchanges in DNA. One such change is depurination, theloss of a purine base from a nucleotide. Depurinationresults when the covalent bond connecting the purine tothe 1�-carbon atom of the deoxyribose sugar breaks ( FIGURE 17.16a), producing an apurinic site—a nucleo-tide that lacks its purine base. An apurinic site cannotact as a template for a complementary base in replication.In the absence of base-pairing constraints, an incorrectnucleotide (most often adenine) is incorporated intothe newly synthesized DNA strand opposite the apurinic

◗

site ( FIGURE 17.16b), frequently leading to an incorporatederror. The incorporated error is then transformed intoa replication error at the next round of replication. Depuri-nation is a common cause of spontaneous mutation; amammalian cell in culture loses approximately 10,000purines every day.

Another spontaneously occurring chemical change thattakes place in DNA is deamination, the loss of an aminogroup (NH2) from a base. Deamination may occur sponta-neously or be induced by mutagenic chemicals.

◗

17.14 Insertions and deletions may result fromstrand slippage.◗

5’5’3’

3’ATGCCTGACTTTTTGCGAAGTACGGACTGAAAA

5’ 3’ACGGACTGAA AA5’5’3’

3’TGCCTGACTT TTTGCGAA

5’5’3’3’ACGGACTGAA AACGCTT5’

5’3’3’

TGCCTGACTT TTTGCGAA

ACGGACTGAA AA

ACGGACTGAA AAACGCTTA

TGCCTGACTT TTGCGAAT

5’3’ TGCCTGACTT TTGCGAAT

Template strandNewly synthesized strand

1 Newly synthesizedstrand loops out,…

3 Template strandloops out,…

4 …resulting in theomission of onenucleotide on thenew strand.

2 …resulting in theaddition of onenucleotide on the new strand.

AATTAATTTTAATTAA

AATTAATTTTAATTAA

AATTAATTTTAATTAA

AATTTTAA

AATTTTAA

AATTTTAA

AATTAATTAATTTTAATTAATTAA

Unequal crossing over

1 If homologouschromosomesmisalign duringcrossing over,…

2 …one crossoverproduct containsan insertion…

3 …and the otherhas a deletion.

17.15 Unequal crossing over produces insertionsand deletions.◗

17.16 Depurination, loss of a purine base from the nucleotide,produces an apurinic site.◗

OH

T

G

G

5’

3’

TGGC T GC

T GC

ACC G ACC G

G

ACCGTGG C

AACGT G C

AACGT G C

ACC G

T GC

AACGAACGTTG C

(a) (b)

Apurinicsite

Strandseparation

Depurination

Strandseparation

Replication

Replication

Templatestrands

Mutant

Normal DNA molecule(no mutation)

DNAsugar–phosphatebackbone

Bases

Pyrimidine

Purine

DNA

1 During replication, the apurinicsite cannot provide a templatefor a complementary base onthe newly synthesized strand.

2 A nucleotide with theincorrect base (most oftenA) is incorporated into thenewly synthesized strand.

3 At the next round ofreplication, this incorrectlyincorporated base will beused as a template,…

5 A nucleotide is incorporatedinto the newly synthesizedstrand opposite the apurinic site.

4 …leading toa permamentmutation.


Deamination may alter the pairing properties of a base:the deamination of cytosine, for example, produces uracil( FIGURE 17.17a), which pairs with adenine during replica-tion. After another round of replication, the adenine willpair with thymine, creating a T�A pair in place of the origi-nal C�G pair (C�G:U�A:T�A); this chemical change is atransition mutation. This type of mutation is usuallyrepaired by enzymes that remove uracil whenever it isfound in DNA. The ability to recognize the product of cyto-sine deamination may explain why thymine, not uracil, isfound in DNA. Some cytosine bases in DNA are naturallymethylated and exist in the form of 5-methylcytosine(5mC; see p. 000 in Chapter 10 and Figure 10.19), whichwhen deaminated becomes thymine ( FIGURE 17.17b).Because thymine pairs with adenine in replication, thedeamination of 5-methylcytosine changes an original C�Gpair to T�A (C�G:5mC�A:T�A). This change cannot bedetected by DNA repair systems, because it produces a nor-mal base. Consequently, C�G:T�A transitions occur fre-quently in eukaryotic cells.

◗

◗

Nazi Germany, Auerbach immigrated to Britain, where sheconducted research on the development of mutants inDrosophila. There she met Herman Muller, who had shownthat radiation induces mutations; he suggested that Auer-bach try to obtain mutants by treating Drosophila withchemicals. Her initial attempts met with little success. Otherscientists were conducting top-secret research on mustardgas (used as a chemical weapon in World War I) andnoticed that it produced many of the same effects as radia-tion. Auerbach was asked to determine whether mustard gaswas mutagenic.

Collaborating with pharmacologist J. M. Robson,Auerbach studied the effects of mustard gas on Drosophilamelanogaster. The experimental conditions were crude.They heated liquid mustard gas over a Bunsen burner onthe roof of the pharmacology building, and the flies wereexposed to the gas in a large chamber. After developing se-rious burns on her hands from the gas, Auerbach let oth-ers carry out the exposures, and she analyzed the flies.Auerbach and Robson showed that mustard gas is indeed apowerful mutagen, reducing the viability of gametes andincreasing the numbers of mutations seen in the offspringof exposed flies. Because the research was part of the secretwar effort, publication of their findings was delayed until1947.

A brief history of HermanMuller

Base analogs One class of chemical mutagens consists ofbase analogs, chemicals with structures similar to that ofany of the four standard bases of DNA. DNA polymerasescannot distinguish these analogs from the standard bases;so, if base analogs are present during replication, they maybe incorporated into newly synthesized DNA molecules. Forexample, 5-bromouracil (5BU) is an analog of thymine; ithas the same structure as that of thymine except that it hasa bromine (Br) atom on the 5-carbon atom instead of amethyl group ( FIGURE 17.18a). Normally, 5-bromouracilpairs with adenine just as thymine does, but it occasionallymispairs with guanine ( FIGURE 17.18b), leading to atransition (T�A:5BU�A:5BU�G:C�G), as shown in

◗

◗

O

CN

NH2

N O

O

UN

H3C H3CN

O

CN

NH2

N O

O

TN

N

H

H

H H H

H H HDeamination Deamination

Cytosine Uracil

(a)

Thymine

(b)

5-Methylcytosine(5mC)

17.17 Deamination alters DNA bases.◗

ConceptsSome mutations arise from spontaneousalterations to DNA structure, such as depurinationand deamination, which may alter the pairingproperties of the bases and cause errors insubsequent rounds of replication.

Chemically Induced MutationsAlthough many mutations arise spontaneously, a number ofenvironmental agents are capable of damaging DNA,including certain chemicals and radiation. Any environ-mental agent that significantly increases the rate of muta-tion above the spontaneous rate is called a mutagen.

The first discovery of a chemical mutagen was made byCharlotte Auerbach, who was born in Germany to a Jewishfamily in 1899. After attending university in Berlin anddoing research, she spent several years teaching at variousschools in Berlin. Faced with increasing anti-Semitism in


488 Chapter 17

FIGURE 17.19. Through mispairing, 5-bromouracil mayalso be incorporated into a newly synthesized DNA strandopposite guanine. In the next round of replication, 5-bro-mouracil may pair with adenine, leading to another transi-tion (G�C:G�5BU:A�5BU:A�T).

Another mutagenic chemical is 2-aminopurine (2AP),which is a base analog of adenine ( FIGURE 17.20). Nor-mally, 2-aminopurine base pairs with thymine, but it maymispair with cytosine, causing a transition mutation(T�A:T�2AP:C�2AP:C�G). Alternatively, 2-amino-purine may be incorporated through mispairing into thenewly synthesized DNA opposite cytosine and later pairwith thymine, leading to a C�G:C�2AP:T�2AP:T�Atransition.

Thus, both 5-bromouracil and 2-aminopurine can pro-duce transition mutations. In the laboratory, mutations by

◗

◗ base analogs can be reversed by treatment with the sameanalog or by treatment with a different analog.

Alkylating agents Alkylating agents are chemicals thatdonate alkyl groups. These agents include methyl (CH3)and ethyl (CH3 – CH2) groups, which are added tonucleotide bases by some chemicals. For example, ethyl-methanesulfonate (EMS) adds an ethyl group to guanine,producing 6-ethylguanine, which pairs with thymine (see Figure 17.20a). Thus, EMS produces C�G:T�A transi-tions. EMS is also capable of adding an ethyl group tothymine, producing 4-ethylthymine, which then pairs withguanine, leading to a T�A:C�G transition. Because EMSproduces both C�G:T�A and T�A:C�G transitions, muta-tions produced by EMS can be reversed by additional treat-ment with EMS. Mustard gas is another alkylating agent.

TN

N

H3C O

O

BuN

N

Br O

O

UN

N

Br O

O

G N

O

N

H

N

A

N

N

H

N

H

H

BuN

N

Br O

O

HH

H

H

–H HHHH NN

N

H H H

Normal pairing Mispairing

Thymine

Normal base

5-Bromouracil

Base analog

(a) (b)

5-Bromouracil Adenine Guanine5-Bromouracil (ionized)

5’3’CTG

GAC5’

3’5’

GAC

3’

5’3’5’

3’

5’3’5’

3’

5’3’5’

3’

5’3’5’

3’

5’3’5’

3’

5’3’5’3’5’

3’5’

3’5’

3’

5’3’

5’3’

CTGGACCTG

CTGGAC

CCGGGCGAC

GAC

GGC

GGC

GAC

CBG

CBG CBG

CBG CBG

Incorporatederror

Replicatederror

Mutant

Strandseparation

Strandseparation

Strandseparation

Replication

Replication

Replication

Conclusion: Incorporation of bromouracil followed bymispairing leads to a TA CG transition mutation.

1 In replication, 5-bromouracil maybecome incorporated into DNA in place ofthymine, producing an incorporated error.

3 In the next replication, this guaninenucleotide pairs with cytosine,leading to a permanent mutation.

2 5-Bromouracil may mispairwith guanine in the nextround of replication.

4 If 5-bromouracil pairswith adenine, noreplication error occurs.

17.19 5-Bromouracil can lead to a replicated error.◗

17.18 5-Bromouracil (a base analog) resembles thymine, exceptthat it has a bromine atom in place of a methyl group on the5-carbon atom. Because of the similarity in their structures, 5-bromouracilmay be incorporated into DNA in place of thymine. Like thymine,5-bromouracil normally pairs with adenine but, when ionized, it may pairwith guanine through wobble.

◗


Deamination In addition to its spontaneous occurrence(see Figure 17.17), deamination can be induced by somechemicals. For instance, nitrous acid deaminates cytosine,creating uracil, which in the next round of replication pairswith adenine (see Figure 17.20b), producing a C�G:T�Atransition mutation. Nitrous acid changes adenine intohypoxanthine, which pairs with cytosine, leading to aT�A:C�G transition. Nitrous acid also deaminates gua-nine, producing xanthine, which pairs with cytosine just asguanine does; however xanthine may also pair withthymine, leading to a C�G:T�A transition. Nitrous acidproduces exclusively transition mutations and, because bothC�G:T�A and T�A:C�G transitions are produced, thesemutations can be reversed with nitrous acid.

Hydroxylamine Hydroxylamine is a very specific base-modifying mutagen that adds a hydroxyl group to cytosine,converting it into hydroxylaminocytosine (see Figure 17.20c).This conversion increases the frequency of a rare tautomer

that pairs with adenine instead of guanine and leads toC�G:T�A transitions. Because hydroxylamine acts onlyon cytosine, it will not generate T�A:C�G transitions;thus, hydroxylamine will not reverse the mutations that itproduces.

Oxidative reactions Reactive forms of oxygen (includingsuperoxide radicals, hydrogen peroxide, and hydroxyl radi-cals) are produced in the course of normal aerobic metabo-lism, as well as by radiation, ozone, peroxides, and certaindrugs. These reactive forms of oxygen damage DNA andinduce mutations by bringing about chemical changesto DNA. For example, oxidation converts guanine into8-oxy-7,8-dihydrodeoxyguanine ( FIGURE 17.21), whichfrequently mispairs with adenine instead of cytosine, caus-ing a G�C:T�A transversion mutation.

Intercalating agents Intercalating agents, such as pro-flavin, acridine orange, ethidium bromide, and dioxin are

◗

17.20 Chemicals may alter DNA bases. (a) The alkylating agentethylmethanesulfonate (EMS) adds an ethyl group to guanine, producing6-ethylguanine, which pairs with thymine, producing a C�G:T�A transitionmutation. (b) Nitrous acid deaminates cytosine to produce uracil, whichpairs with adenine, producing a C�G:T�A transition mutation.(c) Hydroxylamine converts cytosine into hydroxylaminocytosine, whichfrequently pairs with adenine, leading to a C�G:T�A transition mutation.

◗

G H

O

H

H

N

O

N

CN

N

O

A

N

N

H

N

H

HH

N

N

NNN

6

1

31

H3C CH2

T

O

O

N

N

CH3

H

O

H

H

N

N

N H

N

N

NH2

CN

N

O

NH2

UN

N

A

N

N

H

N

H

HH

N

O

O

HO

H

6

14

31

31 3

1H

H

H

H

H

H

N

N

H

H

N

N

H

H

H

Original base

(a)

(b)

(c)

Mutagen Modified base Pairing partner Type ofmutation

CG TA

CG TA

EMS

Guanine O6-Ethylguanine Thymine

Cytosine Uracil Adenine

Cytosine Hydroxylamino-cytosine

Adenine

Nitrous acid(HNO2)

Hydroxylamine(NH2OH) CG TA

490 Chapter 17

about the same size as a nucleotide ( FIGURE 17.22a).They produce mutations by sandwiching themselves(intercalating) between adjacent bases in DNA, distortingthe three-dimensional structure of the helix and causing sin-gle-nucleotide insertions and deletions in replication ( FIGURE 17.22b). These insertions and deletions frequentlyproduce frameshift mutations (which change all amino acidsdownstream of the mutation), and so the mutagenic effectsof intercalating agents are often severe. Because intercalatingagents generate both additions and deletions, they canreverse the effects of their own mutations.

◗

◗

H

G

OH

HN

N

N O

H

H

H

NH

H

N

N

N

NO

N N

Guanine

Oxidativeradicals

8-Oxy-7,8-dihydrodeoxyguanine(may mispair with adenine)

17.21 Oxidative radicals convert guanine into8-oxy-7,8-dihydrodeoxyguanine, which frequentlymispairs with adenine instead of cytosine,producing a C�G:T�A transversion.

◗

ConceptsChemicals can produce mutations by a number ofmechanisms. Base analogs are inserted into DNAand frequently pair with the wrong base.Alkylating agents, deaminating chemicals,hydroxylamine, and oxidative radicals change thestructure of DNA bases, thereby altering theirpairing properties. Intercalating agents wedgebetween the bases and cause single-baseinsertions and deletions in replication.

H2N NH2N

N NNH3C

CH3

CH3

CH3

H

H

H H

H

HH

H

H

H H

H

HH

(a) (b)

Nitrogenousbases

Intercalatedmolecule

Proflavin

Acridine orange

17.22 Intercalating agents such as proflavin andacridine orange insert themselves between adjacentbases in DNA, distorting the three-dimensionalstructure of the helix and causing single-nucleotideinsertions and deletions in replication.

◗

RadiationIn 1927, Herman Muller demonstrated that mutations infruit flies could be induced by X-rays. The results of subse-quent studies showed that X-rays greatly increase muta-tion rates in all organisms. The high energies of X-rays,gamma rays, and cosmic rays ( FIGURE 17.23) are all ca-pable of penetrating tissues and damaging DNA. Theseforms of radiation, called ionizing radiation, dislodge elec-trons from the atoms that they encounter, changing stablemolecules into free radicals and reactive ions, which thenalter the structures of bases and break phosphodiester

◗

X-rays

Cosmic rays/Gamma rays

Ultraviolet (UV)

Infrared (IR)

Microwaves/Radio waves

Visible light

1

10

102

103

104

105

106

Wavelength (nm)

700

400

600

500

Violet

Blue

Blue green

Green

Yellow green

Yellow

Orange

Red

Shorter wavelengths aremore energetic.

Longer wavelengths areless energetic.

17.23 In the electromagnetic spectrum, aswavelength decreases, energy increases. (Adapted from Life 6e, figure 8.5).

◗


bonds in DNA. Ionizing radiation also frequently resultsin double-strand breaks in DNA. Attempts to repair thesebreaks can produce chromosome mutations (discussed inChapter 9).

Ultraviolet light has less energy than that of ionizingradiation and does not eject electrons and cause ionizationbut is nevertheless highly mutagenic. Purine and pyrimi-dine bases readily absorb UV light, resulting in the forma-tion of chemical bonds between adjacent pyrimidinemolecules on the same strand of DNA and in the creationof structures called pyrimidine dimers ( FIGURE 17.24a).Pyrimidine dimers consisting of two thymine bases (calledthymine dimers) are most frequent, but cytosine dimersand thymine – cytosine dimers also can form. These dimersdistort the configuration of DNA ( FIGURE 17.24b) andoften block replication. Most pyrimidine dimers are imme-diately repaired by mechanisms discussed later in thischapter, but some escape repair and inhibit replication andtranscription.

When pyrimidine dimers block replication, cell divi-sion is inhibited and the cell usually dies; for this reason,UV light kills bacteria and is an effective sterilizing agent.For a mutation — a hereditary error in the genetic in-structions — to occur, the replication block must be over-come. How do bacteria and other organisms replicate de-spite the presence of thymine dimers?

Bacteria can circumvent replication blocks producedby pyrimidine dimers and other types of DNA damage bymeans of the SOS system. This system allows replicationblocks to be overcome, but in the process makes numer-ous mistakes and greatly increases the rate of mutation.Indeed, the very reason that replication can proceed inthe presence of a block is that the enzymes in the SOS sys-tem do not strictly adhere to the base-pairing rules. Thetrade-off is that replication may continue and the cell

◗

◗

survives, but only by sacrificing the normal accuracy ofDNA synthesis.

The SOS system is complex, including the products of atleast 25 genes. A protein called RecA binds to the damagedDNA at the blocked replication fork and becomes activated.This activation promotes the binding of a protein calledLexA, which is a repressor of the SOS system. The activatedRecA complex induces LexA to undergo self-cleavage, de-stroying its repressive activity. This inactivation enablesother SOS genes to be expressed, and the products of thesegenes allow replication of the damaged DNA to proceed. TheSOS system allows bases to be inserted into a new DNA strandin the absence of bases on the template strand, but these inser-tions result in numerous errors in the base sequence.

Eukaryotic cells have a specialized DNA polymerasecalled polymerase � (eta) that bypasses pyrimidine dimers.Polymerase � preferentially inserts AA opposite a pyrimi-dine dimer. This strategy seems to be reasonable becauseabout two-thirds of pyrimidine dimers are thymine dimers.However, the insertion of AA opposite a CT dimer results ina C�G:A�T transversion. Polymerase � is therefore said tobe an error-prone polymerase.

P

T

TPP

(a) (b)

AG CATCTCCAAC GTAG

TG GT

Covalentbonds

UV light

Thyminebases

Sugar–phosphatebackbone

5’

3’

5’

3’

2 Thymine dimers distortthe configuration ofthe DNA molecule.

1 UV light causes adjacentthymines to be cross-linkedby covalent bonds.

17.24 Pyrimidine dimers result from Ultraviolet light.(a) Formation of thymine dimer. (b) Distorted DNA.◗

ConceptsIonizing radiation such as X-rays and gamma raysdamage DNA by dislodging electrons from atoms;these electrons then break phosphodiester bondsand alter the structure of bases. Ultraviolet lightcauses mutations primarily by producingpyrimidine dimers that disrupt replication andtranscription. The SOS system enables bacteria toovercome replication blocks but introducesmistakes in replication.

492 Chapter 17

The Study of MutationsBecause mutations often have detrimental effects, they havebeen the subject of intense study by geneticists. These stud-ies have included the analysis of reverse mutations, whichare often sources of important insight into how mutationscause DNA damage; the development of tests to determinethe mutagenic properties of chemical compounds; and theinvestigation of human populations tragically exposed tohigh levels of radiation.

The Analysis of Reverse MutationsThe study of reverse mutations (reversions) can provideuseful information about how mutagens alter DNA struc-ture. For example, any mutagen that produces bothA�T :G�C and G�C:A�T transitions should be able toreverse its own mutations. However, if the mutagen pro-duces only G�C:A�T transitions, then reversion by thesame mutagen is not possible. Hydroxylamine (see Figure17.20c) exhibits this type of one-way mutagenic activity; itcauses G�C:A�T transitions but is incapable of reversingthe mutations that it produces; so we know that it does notproduce A�T :G�C transitions. Ethylmethanesulfonate (seeFigure 17.20a), on the other hand, produces G�C:A�Ttransitions and reverses its own mutations; so we know thatit also produces T�A:C�G transitions.

Analyses of the ability of different mutagens to causereverse mutations can be sources of insight into the molec-ular nature of the mutations. We can use reverse mutationsto determine whether a mutation results from a base substi-tution or a frameshift. Base analogs such as 2-aminopurinecause transitions, and intercalating agents such as acridineorange (see Figure 17.22) produce frameshifts. If a chemicalreverses mutations produced by 2-aminopurine but notthose produced by acridine orange, we can conclude that

the chemical causes transitions and not frameshifts. Ifnitrous acid (which produces both G�C:A�T andA�T:G�C transitions) reverses mutations produced by thechemical but hydroxylamine (which causes only G�C:A�Ttransitions) does not, we know that, like hydroxylamine, thechemical produces only G�C:A�T transitions. Table 17.4illustrates the reverse mutations that are theoretically possi-ble among several mutagenic agents. The actual ability ofmutagens to produce reversals is more complex than sug-gested by Table 17.4 and depends on environmental condi-tions and the organism tested.

Theoretical reverse mutations possible by various mutagenic agentsTable 17.4

TReversal of Mutations by

Ethyl Type of 5-Bromo- 2-Amino- methane Nitrous Hydroxyl- Acridine

Mutagen Mutation uracil purine sulfonate acid amine orange

5-Bromouracil C�G4T�A � � � � �/� �

2-Aminopurine C�G4T�A � � � � �/� �

Nitrous acid C�G4T�A � � � � �/� �

Ethylmethane C�G4T�A � � � � �/� �

sulfonate

Hydroxylamine C�G4T�A � � � � � �

Acridine orange Frameshift � � � � � �

Note: � indicates that reverse mutations occur, � indicates that reverse mutations do not occur, and �/� indicates that only some mutationsare reversed. Not all reverse mutations are equally likely.

ConceptsThe study of the ability of mutagenic agents toproduce reverse mutations provides importantinformation about how mutagens alter DNA.

Detecting Mutations with the Ames TestHumans in industrial societies are surrounded by a multi-tude of artificially produced chemicals: more than 50,000different chemicals are in commercial and industrial usetoday, and from 500 to 1000 new chemicals are introducedeach year. Some of these chemicals are potential carcinogensand may cause potential harm to humans. How can wedetermine which chemicals are hazardous? In a fewinstances, previous human exposure to a specific chemical iscorrelated with an increase in cancer incidence, providinggood evidence that the chemical is a carcinogen. But, ide-ally, we would like to know which chemicals are hazardousbefore we are exposed to them. One method for testing thecancer-causing potential of chemicals is to administer themto laboratory animals (rats or mice) and compare the inci-


dence of cancer in the treated animals with that of controlanimals. These tests are unfortunately time consuming andexpensive. Furthermore, the ability of a substance to causecancer in rodents is not always indicative of its effect onhumans. After all, we aren’t rats!

In 1974, Bruce Ames developed a simple test for evalu-ating the potential of chemicals to cause cancer. The Amestest is based on the principle that both cancer and mutationsresult from damage to DNA, and the results of experimentshave demonstrated that 90% of known carcinogens are alsomutagens. Ames proposed that mutagenesis in bacteriacould serve as an indicator of carcinogenesis in humans.

The Ames test uses four strains of the bacterium Sal-monella typhimurium that have defects in the lipopolysac-charide coat, which normally protects the bacteria fromchemicals in the environment. Furthermore, their DNArepair system has been inactivated, enhancing their suscep-tibility to mutagens.

One of the four strains used in the Ames test detectsbase-pair substitutions; the other three detect differenttypes of frameshift mutations. Each strain carries a muta-tion that renders it unable to synthesize the amino acid his-tidine (his�), and the bacteria are plated onto medium thatlacks histidine ( FIGURE 17.25). Only bacteria that haveundergone a reverse mutation of the histidine gene(his� :his�) are able to synthesize histidine and grow onthe medium. Different dilutions of a chemical to be testedare added to plates inoculated with the bacteria, and thenumber of mutant bacterial colonies that appear on eachplate is compared with the number that appear on controlplates with no chemical (arose through spontaneous muta-tion). Any chemical that significantly increases the numberof colonies appearing on a treated plate is mutagenic and isprobably also carcinogenic.

Some compounds are not active carcinogens but maybe converted into cancer-causing compounds in the body.To make the Ames test sensitive for such potential carcino-gens, a compound to be tested is first incubated in mam-malian liver extract that contains metabolic enzymes. TheAmes test has been applied to thousands of chemicals andcommercial products. An early demonstration of its useful-ness was the discovery, in 1975, that most hair dyes sold inthe United States contained compounds that were muta-genic to bacteria. These compounds were then removedfrom most hair dyes.

◗

More information on the Amestest

Radiation Exposure in HumansPeople are routinely exposed to low levels of radiation fromcosmic, medical, and environmental sources, but there havealso been tragic events that produced exposures of muchhigher degree.

ConceptsThe Ames test uses his� strains of bacteria to testchemicals for their ability to produce his� :his�

mutations. Because mutagenic activity andcarcinogenic potential are closely correlated, theAmes test is widely used to screen chemicals fortheir cancer-causing potential.

17.25 The Ames test is used to identifychemical mutagens.◗


Incubate Incubate

Control plate(no chemical)

Treatment plate(chemical added)

his– bacteria

1 Bacterial his– strains aremixed with liver enzymes(which have the ability toconvert compounds intopotential mutagens).

2 Some of the bacterialstrains are also mixedwith the chemical tobe tested for mutagenic activity.

3 The bacteria arethen plated onmedium thatlacks histidine.

4 Bacterial colonies thatappear on the plateshave undergone ahis– his+ mutation.

5 Any chemical that significantly increases the numberof colonies appearing on the plate is mutagenic andtherefore probably also carcinogenic.

494 Chapter 17

On August 6, 1945, a high-flying American planedropped a single atomic bomb on the city of Hiroshima,Japan. The explosion devastated 4.5 square miles of the city,killed from 90,000 to 140,000 people, and injured almost asmany ( FIGURE 17.26). Three days later, the United Statesdropped an atomic bomb on the city of Nagasaki, this timedestroying 1.5 square miles of city and killing between60,000 and 80,000 people. Huge amounts of radiation werereleased during these explosions and many people wereexposed.

After the war, a joint Japanese – U.S. effort was made tostudy the biological effects of radiation exposure on the sur-vivors of the atomic blasts and their children. Somaticmutations were examined by studying radiation sicknessand cancer among the survivors; germ-line mutations wereassessed by looking at birth defects, chromosome abnor-malities, and gene mutations in children born to people thathad been exposed to radiation.

Geneticist James Neel and his colleagues examinedalmost 19,000 children of parents who were within 2000meters of the center of the atomic blast at Hiroshima orNagasaki, along with a similar number of children whoseparents did not receive radiation exposure. Radiation doseswere estimated for the child’s parents on the basis of carefulassessment of the parents’ location, posture, and position atthe time of the blast. A blood sample was collected fromeach child, and gel electrophoresis was used to investigateamino acid substitutions in 28 proteins. When rare variantswere detected, blood samples from the child’s parents alsowere analyzed to establish whether the variant was inheritedor a new mutation.

Of a total of 289,868 genes examined by Neel and hiscolleagues, only one mutation was found in the childrenof exposed parents; no mutations were found in the con-trol group. From these findings, a mutation rate of3.4 � 10�6 was estimated for the children whose parents

◗

were exposed to the blast, which is within the range ofspontaneous mutation rates observed for other eukary-otes. Neel and his colleagues also examined the frequencyof chromosome mutations, sex ratios of children born toexposed parents, and frequencies of chromosome aneu-ploidy. There was no evidence in any of these assays forincreased mutations among the children of the peoplewho were exposed to radiation from the atomic explo-sions, suggesting that germ-line mutations were notelevated.

Animal studies clearly show that radiation causesgerm-line mutations; so why was there no apparent increasein germ-line mutations among the inhabitants ofHiroshima and Nagasaki? The exposed parents did exhibitan increased incidence of leukemia and other types of can-cers; so somatic mutations were clearly induced. The answerto the question is not known, but the lack of germ-linemutations may be due to the fact that those persons whoreceived the largest radiation doses died soon after theblasts.

Information on studies of thehealth effects of the nuclear blasts at Hiroshima andNagasaki

The Techa River in southern Russia is another placewhere people have been tragically exposed to high levels ofradiation. The Mayak nuclear facility, located 60 miles fromthe city of Chelyabinsk, produced plutonium for nuclearwarheads in the early days of the Cold War. Between 1949and 1956, this plant dumped some 76 million cubic metersof radioactive sludge into the Techa River. People down-stream used the river for drinking water and crop irrigation;some received radiation doses 1700 times the annualamount considered safe by today’s standards. Radiation inthe area was further elevated by a series of nuclear accidents

17.26 Hiroshima was destroyed byan atomic bomb on August 6, 1945.The atomic explosion produced manysomatic mutations among the survivors.(Stanley Troutman/AP.)

◗



at the Mayak plant; the worst was an explosion of a radioac-tive liquid storage tank in 1957, which showered radiationover a 27,000-square-kilometer area.

Although Soviet authorities suppressed informationabout the radiation problems along the Techa until the1990s, Russian physicians lead by Mira Kossenko quietlybegan studying cancer and other radiation-related illnessesamong the inhabitants in the 1960s. They found that theoverall incidence of cancer was elevated among people wholived on the banks of the Techa River.

Most data on radiation exposure in humans are fromthe intensive study of the survivors of the atomic bombingof Hiroshima and Nagasaki. However, the inhabitants ofHiroshima and Nagasaki were exposed in one intense burstof radiation, and these data may not be appropriate forunderstanding the effects of long-term low-dose radiation.Today, U.S. and Russian scientists are studying the people ofthe Techa River region, as well as those exposed to radiationin the Chernobyl accident (see the story at the beginning ofthis chapter), in an attempt to better understand the effectsof chronic radiation exposure on human populations.

DNA RepairThe integrity of DNA is under constant assault from radia-tion, chemical mutagens, and spontaneously arisingchanges. In spite of this onslaught of damaging agents, therate of mutation remains remarkably low, thanks to the effi-ciency with which DNA is repaired. It has been estimatedthat fewer than one in a thousand DNA lesions becomes amutation; all the others are corrected.

There are a number of complex pathways for repairingDNA, but several general statements can be made aboutDNA repair. First, most DNA repair mechanisms requiretwo nucleotide strands of DNA because most replace wholenucleotides, and a template strand is needed to specify thebase sequence. The complementary, double-stranded natureof DNA not only provides stability and efficiency of replica-tion, but also enables either strand to provide the informa-tion necessary for correcting the other.

A second general feature of DNA repair is redundancy,meaning that many types of DNA damage can be correctedby more than one pathway of repair. This redundancytestifies to the extreme importance of DNA repair to thesurvival of the cell: it ensures that almost all mistakes arecorrected. If a mistake escapes one repair system, it’s likelyto be repaired by another system.

We will consider four general mechanisms of DNArepair: mismatch repair, direct repair, base-excision repair,and nucleotide-excision repair (Table 17.5).

Mismatch RepairReplication is extremely accurate: each new copy of DNAhas only one error per billion nucleotides. However, in theprocess of replication, mismatched bases are incorporated

into the new DNA with a frequency of about 10�4 to 10�5;so most of the errors that initially arise are corrected andnever become permanent mutations. Some of these correc-tions are made in proofreading (see p. 000 in Chapter 12).DNA polymerases have the capacity to recognize and cor-rect mismatched nucleotides. When a mismatchednucleotide is added to a newly synthesized DNA strand, thepolymerase stalls. It then uses its 3�:5� exonuclease activ-ity to back up and remove the incorrectly insertednucleotide before continuing with 5�:3� polymerization.

Many incorrectly inserted nucleotides that escapedetection by proofreading are corrected by mismatch repair(see p. 000 in Chapter 12). Incorrectly paired bases distortthe three-dimensional structure of DNA, and mismatch-repair enzymes detect these distortions. In addition todetecting incorrectly paired bases, the mismatch-repair sys-tem corrects small unpaired loops in the DNA, such as thosecaused by strand slippage in replication (see Figure 17.14).Some trinucleotide repeats may form secondary structureson the unpaired strand (see Figure 17.6d), allowing them toescape detection by the mismatch-repair system.

After the incorporation error has been recognized,mismatch-repair enzymes cut out the distorted section ofthe newly synthesized strand and fill the gap with newnucleotides, by using the original DNA strand as a template.For this strategy to work, mismatch repair must have someway of distinguishing between the old and the new strandsof the DNA so that the incorporation error, and not part ofthe original strand, is removed.

The proteins that carry out mismatch repair in E. colidifferentiate between old and new strands by the presenceof methyl groups on special sequences of the old strand.After replication, adenine nucleotides in the sequenceGATC are methylated by an enzyme called Dam methylase.The process of methylation is delayed and so, immediatelyafter replication, the old strand is methylated and the new

Summary of common DNA repairmechanisms

Table 17.5

Repair System Type of Damage Repaired

Mismatch Replication errors, includingmispaired bases and strand slippage

Direct Pyrimidine dimers; other specific types of alterations

Base-excision Abnormal bases, modified bases, and pyrimidine dimers

Nucleotide-excision DNA damage that distorts thedouble helix, includingabnormal bases, modifiedbases, and pyrimidine dimers

496 Chapter 17

strand is not ( FIGURE 17.27a). In E. coli, the proteinsMutS, MutL, and MutH are required for mismatch repair.MutS binds to the mismatched bases and forms a complexwith MutL and MutH; this complex is thought to bring anunmethylated GATC sequence in close proximity to themismatched bases. MutH nicks the unmethylated strand atthe GATC site ( FIGURE 17.27b), and exonucleases degradethe unmethylated strand from the nick to the mismatchedbases ( FIGURE 17.27c). DNA polymerase and DNA ligasefill in the gap on the unmethylated strand with correctlypaired nucleotides ( FIGURE 17.27d).

Mismatch repair in eukaryotic cells is similar to that inE. coli, except that several proteins are related to MutS andseveral are related to MutL. These proteins functiontogether in different combinations to detect different typesof incorporation errors, such as mispaired bases and smallunpaired loops. Eukaryotic cells do not have any proteinsrelated to E. coli MutH. What enzyme makes the nick ineukaryotic cells is not clear. How the old and new strandsare recognized in eukaryotic cells is not known, because insome eukaryotes, such as yeast and fruit flies, there is nodetectable methylation of DNA.

Direct RepairDirect-repair mechanisms do not replace altered nu-cleotides but instead change them back into their original(correct) structures. One of the best-characterized direct-repair mechanisms is photoreactivation of UV-inducedpyrimidine dimers. E. coli and some eukaryotic cells possessan enzyme called photolyase, which uses energy capturedfrom light to break the covalent bonds that link the pyrim-idines in a dimer.

Direct repair also corrects O6-methylguanine, an alkyla-tion product of guanine that pairs with adenine, producingG�C:T�A transversions. An enzyme called O6-methyl-guanine-DNA methyltransferase removes the methyl groupfrom O6-methylguanine, restoring the base to guanine ( FIGURE 17.28).

Base-Excision RepairIn base-excision repair, modified bases are first excised andthen the entire nucleotide is replaced. The excision of modi-fied bases is catalyzed by a set of enzymes called DNA

◗

◗

◗

◗

◗

glycosylases, each of which recognizes and removes a specifictype of modified base by cleaving the bond that links thatbase to the 1�-carbon atom of deoxyribose ( FIGURE 17.29a).Uracil glycosylase, for example, recognizes and removes uracilproduced by the deamination of cytosine. Other glycosylases

◗

5’

3’

G

(a)

(b)

(c)

(d)

New DNA

Old (template) DNA

Nick

MutL MutH

MutS

Methyl group

Methyl group

Methyl group

Methyl group

GATCG

CTAGT

T

GATCCTAG

AT

GATCCTAG

T

GATCCTAG

MutL, MutS, MutH(mismatch repairproteins)

DNA bases

1 In DNA replication, amismatched base was added to the new strand.

2 Methylation at GATC sequences allows old and newly synthesizednucleotide strands to be differentiated: immediately after replication,the old strand will be methylated but the new strand will not.

3 Protein MutS binds to the mismatchedbases and forms a complex with MutHand MutL; the mismatch is broughtclose to a methylated GATC sequence;and the new strand is identified.

4 Exonucleases remove nucleotideson the new strand between theGATC sequence and the mismatch.

5 DNA polymerase then replaces thenucleotides, correcting the mismatch,and DNA ligase seals the nick in thesugar–phosphate backbone.

17.27 Many incorrectly inserted nucleotides thatescape proofreading are corrected by mismatchrepair.

◗

G N

N

N

N

G

O

N

N

N

N

NH2 NH2

OCH3

CH3O6-Methylguanine

Methyltransferase

Guanine

17.28 Direct repair changes nucleotides backinto their original structures.◗


recognize hypoxanthine, 3-methyladenine, 7-methylguanine,and other modified bases.

After the base has been removed, an enzyme called AP(apurinic or apyrimidinic) endonuclease cuts the phospho-diester bond, and other enzymes remove the deoxyribosesugar ( FIGURE 17.29b). DNA polymerase then adds newnucleotides to the exposed 3�-OH group ( FIGURE 17.29c),replacing a section of nucleotides on the damaged strand.The nick in the phosphodiester backbone is sealed by DNAligase ( FIGURE 17.29d), and the original intact sequence isrestored ( FIGURE 17.29e).

Nucleotide-Excision RepairThe final repair pathway that we’ll consider is nucleotide-excision repair, which removes bulky DNA lesions that dis-tort the double helix, such as pyrimidine dimers or largehydrocarbons attached to the DNA. Nucleotide-excisionrepair is quite versatile and can repair many different typesof DNA damage. It is found in cells of all organisms frombacteria to humans and is one of the most important of allrepair mechanisms.

The process of nucleotide excision is complex; inhumans, a large number of genes take part. First, a complexof enzymes scans DNA, looking for distortions of its three-dimensional configuration ( FIGURE 17.30a and b). Whena distortion is detected, additional enzymes separate thetwo nucleotide strands at the damaged region, and sin-gle-strand-binding proteins stabilize the separated strands( FIGURE 17.30c). Next, the sugar – phosphate backboneof the damaged strand is cleaved on both sides of thedamage. One cut is made 5 nucleotides upstream (onthe 3� side) of the damage, and the other cut is made8 nucleotides (in prokaryotes) or from 21 to 23 nucleotides(in eukaryotes) downstream (on the 5� side) of the damage ( FIGURE 17.30d). Part of the damaged strand is peeledaway ( FIGURE 17.30e), and the gap is filled in by DNApolymerase and sealed by DNA ligase ( FIGURE 17.30f).

Other Types of DNA RepairThe DNA repair pathways described so far respond to dam-age that is limited to one strand of a DNA molecule, leavingthe other strand to be used as a template for the synthesis ofnew DNA during the repair process. Some types of DNAdamage, however, affect both strands of the molecule andtherefore pose a more severe challenge to the DNA repairmachinery. Ionizing radiation frequently results in double-strand breaks in DNA. The repair of double-strand breaks isfrequently by homologous recombination. Models forhomologous recombination were described in Chapter 12.

Another type of damage that affects both strands is aninterstrand cross-link, which arises when the two strands ofa duplex are connected through covalent bonds. Interstrandcross-links are extremely toxic to cells because they haltreplication. Several drugs commonly used in chemotherapy,including cisplatin, mitomycin C, psoralen, and nitrogen

◗◗

◗

◗

◗

◗◗

◗◗

DNA ligase

PP P P P P P

P PPP PPP

OH

PP P P P P P

P PPP PPP

3’

3’5’

5’

3’

3’5’

5’

PP P P P P P

P PPP PPP3’

5’

5’

PP P

P

P P P

P PPP PPP3’

3’

3’

3’

3’

PP P P P P P

P PPP PPP3’

5’

5’

3’

5’ 5’

5’

5’

(a)

(b)

(c)

(d)

(e)

DNAglycosylase

DNA

AP site

APendonuclease

DNApolymerase

NTPs

Deoxyribose phosphate + dNMPs

New DNA

17.29 Base-excision repair excises modifiedbases and then replaces the entire nucleotide.◗

498 Chapter 17

mustard, cause interstrand cross-links. Nitrogen mustard,which is structurally related to the mustard gas used byCharlotte Auerbach to induce mutations in Drosophila, wasthe first chemical agent to be used in chemotherapy treat-

(a)

(b)

(c)

(d)

(e)

(f)

Damaged DNA

DNApolymerase,ligase

New DNA

5’3’

1 Damage to the DNA,distorts the configurationof the molecule.

2 An enzyme complexrecognizes the distortionresulting from damage.

3 The DNA is separated,and single-strand-binding proteins stabilizethe single strands.

4 An enzyme cleavesthe strand on bothsides of the damage.

5 A part of thedamaged strandis removed,…

6 …and the gap is filled inby DNA polymerase andsealed by DNA ligase.

17.30 Nucleotide-excision repair consistsof four steps: detection of damage, excisionof damage, polymerization, and ligation.

◗

ment. Little is known about how interstrand cross-links arerepaired. One model proposes that double-strand breaks aremade on each side of the cross-link and are subsequentlyrepaired by the pathways that repair double-strand breaks.

The Basic Pathway of DNA Repair

We have now examined several different mechanisms ofDNA repair. What do these methods have in common?How are they different? Most methods of DNA repairdepend on the presence of two strands, becausenucleotides in the damaged area are removed andreplaced. Nucleotides are replaced in mismatch repair,base excision repair, and nucleotide-excision repair, butare not replaced by direct-repair mechanisms.

Repair mechanisms that include nucleotide removalutilize a common four-step pathway:

1. Detection: The damaged section of the DNA isrecognized.

2. Excision: DNA repair endonucleases nick thephosphodiester backbone on one or both sides of theDNA damage.

3. Polymerization: DNA polymerase adds nucleotides tothe newly exposed 3�-OH group by using the otherstrand as a template and replacing damaged (andfrequently some undamaged) nucleotides.

4. Ligation: DNA ligase seals the nicks in the sugar –phosphate backbone.

The primary differences in the mechanisms of mis-match, base-excision, and nucleotide-excision repair are inthe details of detection and excision. In base-excision andmismatch repair, a single nick is made in the sugar–phos-phate backbone on one side of the damaged strand; innucleotide-excision repair, nicks are made on both sides ofthe DNA lesion. In base-excision repair, DNA polymerasedisplaces the old nucleotides as it adds new nucleotides to the3� end of the nick; in mismatch repair, the old nucleotidesare degraded; and, in nucleotide-excision repair, nucleotidesare displaced by helicase enzymes. All three mechanisms useDNA polymerase and ligase to fill in the gap produced by theexcision and removal of damaged nucleotides.

Additional information onDNA repair

Genetic Diseases and Faulty DNA RepairSeveral human diseases are connected to defects in DNArepair. These diseases are often associated with high inci-dences of specific cancers, because defects in DNA repair

Connecting Concepts



lead to increased rates of mutation. This concept is dis-cussed further in Chapter 21.

Among the best studied of the human DNA repair dis-eases is xeroderma pigmentosum ( FIGURE 17.31), a rareautosomal recessive condition that includes abnormal skinpigmentation and acute sensitivity to sunlight. Persons whohave this disease also have a strong predisposition to skincancer, with an incidence from 1000 to 2000 times thatfound in unaffected people.

Sunlight includes a strong UV component; so exposureto sunlight produces pyrimidine dimers in the DNA of skincells. Although human cells lack photolyase (the enzymethat repairs pyrimidine dimers in bacteria), most pyrimi-dine dimers in humans can be corrected by nucleotide-excision repair. However, the cells of most people withxeroderma pigmentosum are defective in nucleotide-excision repair, and many of their pyrimidine dimers gouncorrected and may lead to cancer.

Xeroderma pigmentosum can result from defectsin several different genes; studies have identified at leastseven different xeroderma pigmentosum complementationgroups, meaning that at least seven genes are required fornucleotide-excision repair in humans. Recent molecularresearch has led to the identification of genetic defects ofnucleotide-excision repair associated with these comple-mentation groups. Some persons with xeroderma pigmen-tosum have mutations in a gene encoding the protein thatrecognizes and binds to damaged DNA; others have muta-tions in a gene encoding helicase. Still others have defects inthe genes that play a role in cutting the damaged strand on

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the 5� or 3� sides of the pyrimidine dimer. Some personshave a slightly different form of the disease (xeroderma pig-mentosum variant) owing to mutations in the gene encod-ing polymerase �, the DNA polymerase that bypassespyrimidine dimers by inserting AA.

Two other genetic diseases due to defects in nucleotide-excision repair are Cockayne syndrome and trichothiodys-trophy (also known as brittle-hair syndrome). Persons whohave either of these diseases do not have an increased risk ofcancer but do exhibit multiple developmental and neuro-logical problems. Both diseases result from mutations insome of the same genes that cause xeroderma pigmento-sum. Several of the genes taking part in nucleotide-excisionrepair produce proteins that also play a role in recombina-tion and the initiation of transcription. These other func-tions may account for the developmental symptoms seen inCockayne syndrome and trichothiodystrophy.

Another genetic disease caused by faulty DNA repair isan inherited form of colon cancer called hereditary non-polyposis colon cancer (HNPCC). This cancer is one of themost common hereditary cancers, accounting for about15% of colon cancers. Research indicate that HNPCC arisesfrom mutations in the proteins that carry out mismatchrepair (see Figure 17.27).

Li-Fraumeni syndrome is caused by mutations in agene called p53, which plays an important role in regulatingthe cell cycle. The product encoded by the p53 gene can haltcell division until damage to DNA has been repaired; it canalso directly stimulate DNA repair. The p53 gene productmay actually cause cells with damaged DNA to self-destruct(undergo apoptosis, or controlled cell death; see Chapter21), preventing their mutated genetic instructions frombeing passed on. Patients who have Li-Fraumeni syndromeexhibit multiple independent cancers in different tissues.Some additional genetic diseases associated with defectiveDNA repair are summarized in Table 17.6.

17.31 Xeroderma pigmentosum is a humandisease that results from defects in DNA repair.(Ken Greer/Visuals Unlimited.)

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ConceptsDefects in DNA repair are the underlying cause ofseveral genetic diseases. Many of these diseasesare characterized by a predisposition to cancer.

Additional information aboutxeroderma pigmentosum

This chapter has been our first comprehensive look atmutations, but we have been considering and using muta-tions throughout the book.

Mutation is a fact of life. Our DNA is continuallyassaulted by spontaneously arising and environmentally


Connecting Concepts Across Chapters

500 Chapter 17

induced mutations. These mutations are the raw materialof evolution and, in the long run, allow organisms toadapt to the environment, a topic that will be taken up inChapter 23. In spite of their long-term contribution tospecies evolution, the vast majority of mutations are, inthe short term, detrimental to cells. The fact that mostare detrimental is evidenced by the number mechanismsthat cells possess to reduce the generation of errors inDNA and to repair those that do arise. A dominanttheme of this chapter is that cells go to great lengths toprevent mutations.

This chapter has incorporated information pre-sented in a number of earlier chapters, which you mightwant to review for a better understanding of theprocesses and structures discussed in the current chap-ter. Chromosome mutations and transposable elements(which frequently cause mutations) are discussed inChapters 9 and 11. Although the structural nature ofthese mutations is different from that of gene muta-tions, many fundamental aspects of the mutationalprocess that were introduced in this chapter also applyto these other types of mutations. The study of genemutations is fundamentally about changes in DNA

structure; so the discussion of DNA structure in Chapter10 is critical for understanding the nature of mutationsand how they arise. Some mutations spontaneously arisefrom errors in replication, and many DNA repair mech-anisms include some DNA synthesis; hence, the processof replication outlined in Chapter 12 also is important.The relation between the nucleotide sequences of DNAand the amino acid sequences of proteins, which is dis-cussed in Chapter 15, is particularly relevant for under-standing the phenotypic effects of mutations and thenature of intra- and intergenic suppressors. Some of thematerial covered on bacterial and viral genetics in Chap-ter 15 is helpful for understanding complementationand the Ames test.

The current chapter has provided information thatis important for understanding material presented in fu-ture chapters. Mutation is the molecular basis of cancer;so the contents of the current chapter will be highly rele-vant to the discussion of cancer genetics in Chapter 21.The importance of the mutation process to evolutionwill be revisited in Chapter 23.

Genetic diseases associated with defects in DNA repair systemsTable 17.6

Disease Symptoms Genetic Defect

Xeroderma pigmentosum Frecklelike spots on skin, Defects in sensitivity to sunlight, predis- nucleotide-excision repairposition to skin cancer

Cockayne syndrome Dwarfism, sensitivity to sunlight, Defects in premature aging, deafness, nucleotide-excision repairmental retardation

Trichothiodystrophy Brittle hair, skin abnormalities, Defects in short stature, immature sexual nucleotide-excision repairdevelopment, characteristic facial features

Hereditary nonpolyposis colon cancer Predisposition to colon cancer Defects in mismatch repair

Fanconi anemia Increased skin pigmentation, Possibly defects in the repair abnormalities of skeleton, heart, of interstrand cross-linksand kidneys, predisposition to leukemia

Ataxia telangiectasia Defective muscle coordination, Defects in DNA damagedilation of blood vessels in skin detection and responseand eyes, immune deficiencies, sensitivity to ionizing radiation, predisposition to cancer

Li-Fraumeni syndrome Predisposition to cancer in many Defects in DNA damagedifferent tissues response


• Mutations are heritable changes in genetic information. Theyare important for the study of genetics and can be used tounravel other biological processes.

• Somatic mutations occur in somatic cells; germ-linemutations occur in cells that give rise to gametes. Genemutations are genetic alterations that affect a single gene;chromosome mutations entail changes in the number orstructure of chromosomes.

• The simplest type of mutation is a base substitution, a changein a single base pair of DNA. Transitions are basesubstitutions in which purines are replaced by purines orpyrimidines are replaced by pyrimidines. Transversions arebase substitutions in which a purine replaces a pyrimidine ora pyrimidine replaces a purine.

• Insertions are the addition of nucleotides, and deletions arethe removal of nucleotides; these mutations often change thereading frame of the gene.

• Expanding trinucleotide repeats are mutations in which thenumber of copies of a trinucleotide increases through time;they are responsible for several human genetic diseases.

• A missense mutation alters the coding sequence so that oneamino acid substitutes for another. A nonsense mutationchanges a codon that specifies an amino acid to a terminationcodon. A silent mutation produces a synonymous codon thatspecifies the same amino acid as the original sequence, whereasa neutral mutation alters the amino acid sequence but does notchange the functioning of the protein. A suppressor mutationreverses the effect of a previous mutation at a different site andmay be intragenic (within the same gene as the originalmutation) or intergenic (within a different gene).

• Mutation rate is the frequency with which a particularmutation arises in a population, whereas mutation frequencyis the incidence of a mutation in a population. Mutation ratesare usually low and are influenced by both genetic andenvironmental factors.

• Some mutations occur spontaneously. These mutationsinclude the mispairing of bases in replication andspontaneous depurination and deamination.

• Insertions and deletions may arise from strand slippage inreplication or from unequal crossing over.

• Base analogs may become incorporated into DNA inreplication and pair with the wrong base in subsequentreplication events. Alkylating agents and hydroxylaminemodify the chemical structure of bases and lead tomutations. Intercalating agents insert into the DNA moleculeand cause single-nucleotide additions and deletions.Oxidative reactions alter the chemical structures of bases.

• Ionizing radiation is mutagenic, altering base structures andbreaking phosphodiester bonds. Ultraviolet light producespyrimidine dimers, which block replication. Bacteria use theSOS response to overcome replication blocks produced bypyrimidine dimers and other lesions in DNA, but the SOSresponse causes the occurrence of more replication errors.Pyrimidine dimers in eukaryotic cells can be bypassed byDNA polymerase � but may result in the placement ofincorrect bases opposite the dimer.

• The analysis of reverse mutations provides information aboutthe molecular nature of the original mutation.

• The Ames tests uses bacteria to assess the mutagenic potentialof chemical substances.

• Most damage to DNA is corrected by DNA repairmechanisms. These mechanisms include mismatch repair,direct repair, base-excision repair,nucleotide-excision repair, and other repair pathways.Although the details of the different DNA repair mechanismsvary, most require two strands of DNA and exhibit someoverlap in the types of damage repaired. Proofreading andmismatch repair correct errors that arise in replication.Direct-repair mechanisms change the altered nucleotides backinto their original condition, whereas base-excision andnucleotide-excision repair mechanisms replace nucleotidesaround the damaged segment of the DNA.

• Defects in DNA repair are the underlying cause of severalgenetic diseases.

mutation (p. 000)somatic mutation (p. 000)germ-line mutation (p. 000)gene mutation (p. 000)base substitution (p. 000)transition (p. 000)transversion (p. 000)insertion (p. 000)

deletion (p. 000)frameshift mutation (p. 000)in-frame insertion (p. 000)in-frame deletion (p. 000)expanding trinucleotide repeat

(p. 000)forward mutation (p. 000)

reverse mutation (reversion)(p. 000)

missense mutation (p. 000)nonsense mutation (p. 000)silent mutation (p. 000)neutral mutation (p. 000)loss-of-function mutation

(p. 000)

gain-of-function mutation(p. 000)

conditional mutation (p. 000)lethal mutation (p. 000)suppressor mutation (p. 000)intragenic suppressor

mutation (p. 000)

IMPORTANT TERMS

CONCEPTS SUMMARY

502 Chapter 17

1. A codon that specifies the amino acid Asp undergoes a single-base substitution that yields a codon that specifies Ala. Refer tothe genetic code in Figure 15.12 and give all possible DNAsequences for the original and the mutated codon. Is the mutationa transition or a transversion?

• SolutionThere are two possible RNA codons for Asp: GAU and GAC.The DNA sequences that encode these codons will becomplementary to the RNA codons: CTA and CTG. There arefour possible RNA codons for Ala: GCU, GCC, GCA, and GCG,which correspond to DNA sequences CGA, CGG, CGT, andCGC. If we organize the original and mutated sequences asshown in the following table, it is easy to see what type ofmutations may have occurred:

Possible original sequence Possible mutated sequence for Asp for Ala

CTA CGACTG CGG

CGTCGC

If the mutation is confined to a single-base substitution, thenthe only mutations possible are that CTA mutated to CGA orthat GTG mutated to CGG. In both, there is a T :Gtransversion in the middle nucleotide of the codon.

2. A gene encodes a protein with the following amino acidsequence:

Met-Arg-Cys-Ile-Lys-Arg

A mutation of a single nucleotide alters the amino acid sequenceto:

Met-Asp-Ala-Tyr-Lys-Gly-Glu-Ala-Pro-Val

A second single-nucleotide mutation occurs in the same geneand suppresses the effects of the first mutation (an intragenicsuppressor). With the original mutation and the intragenicsuppressor present, the protein has the following amino acidsequence:

Met-Asp-Gly-Ile-Lys-Arg

What is the nature and location of the first mutation and theintragenic suppressor mutation?

• SolutionThe first mutation alters the reading frame, because all aminoacids after Met are changed. Insertions and deletions affect thereading frame; so the original mutation consists of a single-nucleotide insertion or deletion in the second codon. Theintragenic suppressor restores the reading frame; so theintragenic suppressor also is most likely a single-nucleotideinsertion or deletion: if the first mutation is an insertion, thesuppressor must be a deletion; if the first mutation is a deletion,then the suppressor must be an insertion. Notice that theprotein produced by the suppressor still differs from the originalprotein at the second and third amino acids, but thesuppressor’s second amino acid is the same as that in theprotein produced by the original mutation. Thus the suppressormutation must have occurred in the third codon, because thesuppressor does not alter the second amino acid.

3. The mutations produced by the following compounds arereversed by the substances shown. What conclusions can youmake about the nature of the mutations originally produced bythese compounds?

intergenic suppressormutation (p. 000)

mutation rate (p. 000)mutation frequency (p. 000)spontaneous mutation (p. 000)induced mutation (p. 000)

incorporated error (p. 000)replicated error (p. 000)strand slippage (p. 000)unequal crossing over (p. 000)depurination (p. 000)deamination (p. 000)

mutagen (p. 000)base analog (p. 000)intercalating agent (p. 000)pyrimidine dimer (p. 000)SOS system (p. 000)Ames test (p. 000)

direct repair (p. 000)base-excision repair (p. 000)nucleotide-excision repair

(p. 000)

Worked Problems

• SolutionThe ability of various compounds to produce reverse mutationsreveals important information about the nature of the originalmutation.

(a) Mutations produced by compound 1 are reversed by5-bromouracil, which produces both A�T:G�C and G�C:A�Ttransitions. This tells us that compound 1 produces single-basesubstitutions that may include the generation of either A�T or

Reversed by

Mutations 5-Bromouracil EMS Hydroxy- Acridineproduced lamine orangebycompound

(a) 1 Yes Yes No No(b) 2 Yes Yes Some No(c) 3 No No No Yes(d) 4 Yes Yes Yes Yes


G�C pairs. The mutations produced by compound 1 are alsoreversed by EMS, which, like 5-bromouracil, produces bothA�T:G�C and G�C:A�T transitions; so no additionalinformation is provided here. Hydroxylamine does not reverse themutations produced by compound 1. Because hydroxylamineproduces only C�G:T�A transitions, we know that compound 1does not generate C�G base pairs. Acridine orange, anintercalating agent that produces frameshift mutations, also doesnot reverse the mutations, revealing that compound 1 producesonly single-base-pair substitutions, not insertions or deletions. Insummary, compound 1 appears to causes single-base substitutionsthat generate T�A but not G�C base pairs.

(b) Compound 2 generates mutations that are reversed by5-bromouracil and EMS, indicating that it may produce G�C or

A�T base pairs. Some of these mutations are reversed byhydroxylamine, which produces only C�G:T�A transitions. Thisindicates that some of the mutations produced by compound 2are T�A base pairs. None of the mutations are reversed byacridine orange; so compound 2 does not induce insertions ordeletions. In summary, compound 2 produces single-basesubstitutions that generate both G�C and A�T base pairs.

(c) Compound 3 produces mutations that are reversed only byacridine orange; so compound 3 appears to produce onlyinsertions and deletions.

(d) Compound 4 is reversed by 5 bromouracil, EMS, hydrox-ylamine, and acridine orange, indicating that this compoundproduces single-base substitutions, which include both G�C andA�T base pairs, and insertions and deletions.

MOLECULAR EVOLUTION

This exercise introduces you to some of the basic principles ofmolecular evolution, the study of the ways in which moleculesevolve, and the reconstruction of the evolutionary history of

molecules and organisms. You will use several of the Internettools most frequently used by contemporary molecular geneticiststo analyze analogous sequences from related organisms.

The New GeneticsMINING GENOMES

COMPREHENSION QUESTIONS

1. What is the difference between somatic mutations andgerm-line mutations?

2. What is the difference between a transition and atransversion? Which type of base substitution is usuallymore common? Why?

3. Briefly describe expanding trinucleotide repeats. How dothey account for the phenomenon of anticipation?

4. What is the difference between a missense mutation and anonsense mutation? A silent mutation and a neutralmutation?

5. Briefly describe two different ways that intragenicsuppressors may reverse the effects of mutations.

6. How do intergenic suppressors work?

7. What is the difference between mutation frequency andmutation rate?

8. What is the cause of errors in DNA replication?

9. How do insertions and deletions arise?

10. How do base analogs lead to mutations?

11. How do alkylating agents, nitrous acid, and hydroxylamineproduce mutations?

12. What types of mutations are produced by ionizing and UVradiation?

13. What is the SOS system and how does it lead to an increasein mutations?

14. What is the purpose of the Ames test? How are his�

bacteria used in this test?

15. List at least three different types of DNA repair and brieflyexplain how each is carried out.

16. What features do mismatch repair, base-excision repair, andnucleotide-excision repair have in common?

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1 7. A codon that specifies the amino acid Gly undergoes asingle-base substitution to become a nonsense mutation. Inaccord with the genetic code given in Figure 15.12, is thismutation a transition or a transversion? At which positionof the codon does the mutation occur?

18. (a) If a single transition occurs in a codon that specifies Phe,what amino acids could be specified by the mutated sequence?(b) If a single transversion occurs in a codon that specifiesPhe, what amino acids could be specified by the mutatedsequence?

APPLICATION QUESTIONS AND PROBLEMS

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504 Chapter 17

(c) If a single transition occurs in a codon that specifiesLeu, what amino acids could be specified by the mutatedsequence?

(d) If a single transversion occurs in a codon that specifiesLeu, what amino acids could be specified by the mutatedsequence?

19. Hemoglobin is a complex protein that contains fourpolypeptide chains. The normal hemoglobin found inadults — called adult hemoglobin — consists of two � andtwo � polypeptide chains, which are encoded by differentloci. Sickle-cell hemoglobin, which causes sickle-cell anemia,arises from a mutation in the � chain of adult hemoglobin.Adult hemoglobin and sickle-cell hemoglobin differ in asingle amino acid: the sixth amino acid from one end in adulthemoglobin is glutamic acid, whereas sickle-cell hemoglobinhas valine at this position. After consulting the genetic codeprovided in Figure 15.12, indicate the type and location ofthe mutation that gave rise to sickle-cell anemia.

20. The following nucleotide sequence is found on the templatestrand of DNA. First, determine the amino acids of theprotein encoded by this sequence by using the genetic codeprovided in Figure 15.12. Then, give the altered amino acidsequence of the protein that will be found in each of thefollowing mutations.

(a) Mutant 1: A transition at nucleotide 11.

(b) Mutant 2: A transition at nucleotide 13.

(c) Mutant 3: A one-nucleotide deletion at nucleotide 7.

(d) Mutant 4: A T:A transversion at nucleotide 15.

(e) Mutant 5: An addition of TGG after nucleotide 6.

(f) Mutant 6: A transition at nucleotide 9.

21. A polypeptide has the following amino acid sequence:

Met-Ser-Pro-Arg-Leu-Glu-Gly

The amino acid sequence of this polypeptide was determinedin a series of mutants listed in parts a through e. For eachmutant, indicate the type of change that occurred in theDNA (single-base substitution, insertion, deletion) and thephenotypic effect of the mutation (nonsense mutation,missense mutation, frameshift, etc.).

(a) Mutant 1: Met-Ser-Ser-Arg-Leu-Glu-Gly

(b) Mutant 2: Met-Ser-Pro

(c) Mutant 3: Met-Ser-Pro-Asp-Trp-Arg-Asp-Lys

(d) Mutant 4: Met-Ser-Pro-Glu-Gly

(e) Mutant 5: Met-Ser-Pro-Arg-Leu-Leu-Glu-Gly


Met-Trp-His-Val-Ala-Ser-Phe.

A mutation occurs in the gene. The mutant protein has thefollowing amino acid sequence:

Met-Trp-His-Met-Ala-Ser-Phe.

An intragenic suppressor restores the amino acid sequenceto that of the original the protein:

Met-Trp-His-Arg-Ala-Ser-Phe.

Give at least one example of base changes that couldproduce the original mutation and the intragenicsuppressor? (Consult the genetic code in Figure 15.12.)


Met-Lys-Ser-Pro-Ala-Thr-Pro

A nonsense mutation from a single-base-pairsubstitution occurs in this gene, resulting in a protein withthe amino acid sequence Met-Lys. An intergenic suppressormutation allows the gene to produce the full-length protein.With the original mutation and the intergenic suppressorpresent, the gene now produces a protein with the followingamino acid sequence:

Met-Lys-Cys-Pro-Ala-Thr-Pro

Give the location and nature of the original mutation andthe intergenic suppressor.

24. Can nonsense mutations be reversed by hydroxylamine? Whyor why not?

25. XG syndrome is a rare genetic disease that is due to anautosomal dominant gene. A complete census of a smallEuropean country reveals that 77,536 babies were born in2000, of whom 3 had XG syndrome. In the same year, thiscountry had a population of 5,964,321 people, and there were35 living persons with XG syndrome. What are the mutationrate and mutation frequency of XG syndrome for this country?

26. The following nucleotide sequence is found in a short stretchof DNA:

5�– ATGT – 3�3�– TACA – 5�

If this sequence is treated with hydroxylamine, whatsequences will result after replication?

27. The following nucleotide sequence is found in a short stretchof DNA:

5�– AG – 3�3�– TC – 5�

(a) Give all the mutant sequences that may result fromspontaneous depurination in this stretch of DNA.

(b) Give all the mutant sequences that may result fromspontaneous deamination in this stretch of DNA.

Sequenceof DNA template

3�– TAC TGG CCG TTA GTT GAT ATA ACT – 5�1 24

Nucleotidenumber

↵

↵

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28. In many eukaryotic organisms, a significant proportion ofcytosine bases are naturally methylated to 5-methylcytosine.Through evolutionary time, the proportion of AT base pairsin the DNA of these organisms increases. Can you suggest apossible mechanism by which this increase occurs?

29. A chemist synthesizes four new chemical compounds in thelaboratory and names them PFI1, PFI2, PFI3, and PFI4. Hegives the PFI compounds to a geneticist friend and asks herto determine their mutagenic potential. The geneticist findsthat all four are highly mutagenic. She also tests the capacityof mutations produced by the PFI compounds to be reversedby other known mutagens and obtains the following results.What conclusions can you make about the nature of themutations produced by these compounds?

30. A plant breeder wants to isolate mutants in tomatoes thatare defective in DNA repair. However, this breeder does nothave the expertise or equipment to study enzymes in DNArepair systems. How could the breeder identify tomatoplants that are deficient in DNA repair? What are the traitsto look for?

31. A genetics instructor designs a laboratory experiment tostudy the effects of UV radiation on mutation in bacteria.In the experiment, the students expose bacteria plated onpetri plates to UV light for different lengths of time, placethe plates in an incubator for 48 hours, and then count thenumber of colonies that appear on each plate. The platesthat have received more UV radiation should have morepyrimidine dimers, which block replication; thus, fewercolonies should appear on the plates exposed to UV lightfor longer periods of time. Before the students carry out theexperiment, the instructor warns them that, while thebacteria are in the incubator, the students must not openthe incubator door unless the room is darkened. Whyshould the bacteria not be exposed to light?

Reversed by

Mutations 2-Amino- Nitrous- Hydroxy- Acridineproduced purine acid lamine orangeby

PFI1 Yes Yes Some NoPFI2 No No No NoPFI3 Yes Yes No NoPFI4 No No No Yes

CHALLENGE QUESTIONS

32. Ochre and amber are two types of nonsense mutations.Before the genetic code was worked out, Sydney Brenner,Anthony O. Stretton, and Samuel Kaplan applied differenttypes of mutagens to bacteriophages in an attempt todetermine the bases present in the codons responsible foramber and ochre mutations. They knew that ochre and ambermutants were suppressed by different types of mutations,demonstrating that each was a different termination codon.They obtained the following results.

(1) A single-base substitution could convert an ochremutation into an amber mutation.

(2) Hydroxylamine induced both ochre and amber mutationsin wild-type phages.

(3) 2-Aminopurine caused ochre to mutate to amber.(4) Hydroxylamine did not cause ochre to mutate to amber.

These data do not allow the complete nucleotide sequenceof the amber and ochre codons to be worked out, but theydo provide some information about the bases found in thenonsense mutations.

(a) What conclusions about the bases found in the codonsof amber and ochre mutations can be made from theseobservations?

(b) Of the three nonsense codons (UAA, UAG, UGA),which represents the ochre mutation.

33. To determine whether radiation associated with the atomicbombings of Hiroshima and Nagasaki produced recessivegerm-line mutations, scientists examined the sex ratio of thechildren of the survivors of the blasts. Can you explain whyan increase in germ-line mutations might be expected to alterthe sex ratio?

34. The results of several studies provide evidence that DNArepair is rapid in genes that are undergoing transcription andthat some proteins that play a role in transcription alsoparticipate in DNA repair. How are transcription and DNArepair related? Why might a gene that is being transcribedbe repaired faster than a gene that is not being transcribed?

SUGGESTED READINGS

Balter, M. 1995. Filtering a river of cancer data. Science267:1084–1086.Article describing the nuclear disaster on the Techa river inRussia.

Beale, G. 1993. The discovery of mustard gas mutagenesis byAuerbach and Robson in 1941. Genetics 134:393–399.An informative and personal account of Auerbach’s life andresearch.

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Dovoret, R. 1979. Bacterial tests for potential carcinogens.Scientific American 241(2):40–49.A discussion of the Ames tests and more recent tests ofmutagenesis in bacteria.

Drake, J.W., and R.H. Baltz. 1976. The biochemistry ofmutagenesis. Annual Review of Biochemistry 45:11–37.A discussion of how mutations are produced by mutagenicagents.

Dubrova, Y.E., V.N. Nesterov, N.G. Krouchinsky, V.A. Ostapenko,R. Neumann, D.L. Neil, and A.J. Jeffreys. 1996. Humanminisatellite mutation rate after the Chernobyl accident.Nature 380:683–686.A report of increased germ-line mutation rate in peopleexposed to radiation in the Chernobyl accident.

Goodman, M.F. 1995. DNA models: mutations caught in the act.Nature 378:237–238.A review of the role of tautomerization in replication errors.

Hoeijmakers, J.H., and D. Bootsma. 1994. Incisions for excision.Nature 371:654–655.Commentary on the proteins in eukaryotic nucleotide-excisionrepair.

Martin, J.B. 1993. Molecular genetics of neurological diseases.Science 262:674–676.A discussion of expanding trinucleotide repeats as cause ofneurological diseases.

Modrich, P. 1991. Mechanisms and biological effects ofmismatch repair. Annual Review of Genetics 25:229–253.A comprehensive review of mismatch repair.

Neel, J.V., C. Satoh, H.B. Hamilton, M. Otake, K. Goriki, T.Kageoka, M. Fujita, S. Neriishi, and J. Asakawa. 1980. Searchfor mutations affecting protein structure in children of atomicbomb survivors: preliminary report. Proceedings of the NationalAcademy of Sciences of the United States of America.77:4221–4225.A report of the gene mutations in the children of survivors ofthe atomic bombings in Japan.

Sancar, A. 1994. Mechanisms of DNA excision repair. Science266:1954–1956.An excellent review of research on excision repair. This issue ofScience was about the “molecule of the year” for 1994, whichwas DNA repair (actually not a molecule).

Schull, W.J., M. Otake, and J.V. Neel. 1981. Genetic effects of theatomic bombs: a reappraisal. Science 213:1220–1227.Research findings concerning the genetic effects of radiationexposure in survivors of the atomic bombings in Japan.

Shcherbak, Y.M. 1996. Ten years of the Chernobyl era. ScientificAmerican 274(4):44–49.Considers the long-term effects of the Chernobyl accident.

Sinden, R.R. 1999. Biological implications of DNA structuresassociated with disease-causing triplet repeats. AmericanJournal of Human Genetics 64:346–353.A good summary of disease-causing trinucleotide repeats andsome models for how they might arise.

Tanaka, K., and R.D. Wood. 1994. Xeroderma pigmentosum andnucleotide excision repair. Trends in Biochemical Sciences19:84–86.A review of the molecular basis of xeroderma pigmentosum.

Yu, S., J. Mulley, D. Loesch, G. Turner, A. Donnelly, A. Gedeon,D. Hillen, E. Kremer, M. Lynch, M. Pritchard, G.R. Sunderland,and R.I. Richards. 1992. Fragile-X syndrome: unique geneticsof the heritable unstable element. American Journal of HumanGenetics 50:968–980.A research report describing the expanding trinucleotide repeatthat causes fragile-X syndrome.

17 DNA Repair Gene MMutations aand · analysis of mutations. Finally, we take a look at DNA repair...

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