Mutations, Mutagenesis, Mutations, Mutagenesis, and Repairand Repair

Chapter 10 Chapter 10

The ProblemThe Problem

DNA extremely long, fragileDNA extremely long, fragile Subject to both physical and Subject to both physical and

chemical damagechemical damage Consequences could be lethal Consequences could be lethal

for organism or offspringfor organism or offspring

MutationMutation A heritable change in the base A heritable change in the base

sequence of DNAsequence of DNA Point mutation- change in a single Point mutation- change in a single

base positionbase position AdditionsAdditions DeletionsDeletions SubstitutionsSubstitutions

TransitionsTransitions TransversionsTransversions

Multiple mutationsMultiple mutations

Frameshift mutations}

Consequences of MutationConsequences of Mutation Silent Mutation---base change, no amino acid Silent Mutation---base change, no amino acid

changechange Neutral Mutation--- Base change resulting in aa Neutral Mutation--- Base change resulting in aa

change that does not affect protein functionchange that does not affect protein function EX. Apartic acid (D) EX. Apartic acid (D) Glutamic acid (E)Glutamic acid (E)

Missense mutation---altered codon, new aa with Missense mutation---altered codon, new aa with different chemical properties. Function affected. different chemical properties. Function affected.

Nonsense mutation---base pair substitution Nonsense mutation---base pair substitution results in a stop codon (and shorter results in a stop codon (and shorter polypeptide)polypeptide)

Frameshift mutations—additions or deletions. Frameshift mutations—additions or deletions. Peptide may be longer or shorter.Peptide may be longer or shorter.

Sense mutation?Sense mutation?

Other TermsOther Terms

Conditional Mutation—wild type Conditional Mutation—wild type function except under certain function except under certain ((permissivepermissive) conditions ) conditions Ex. Temperature sensitive mutants show Ex. Temperature sensitive mutants show

mutant phenotype only at certain mutant phenotype only at certain temperaturestemperatures

Leaky mutations— a missense amino Leaky mutations— a missense amino acid change that reduces but doesn’t acid change that reduces but doesn’t eliminate protein functioneliminate protein function

MutagenesisMutagenesis

The process of mutationThe process of mutation Mutagen—anything that Mutagen—anything that

promotes ort causes promotes ort causes mutationsmutations ChemicalChemical PhysicalPhysical

Mutation-CausesMutation-Causes Incorrect base pairing due to Incorrect base pairing due to

tautomeric shiftstautomeric shifts Removal of nitrogenous basesRemoval of nitrogenous bases Alteration of nitrogenous basesAlteration of nitrogenous bases Addition or deletion of nucleotidesAddition or deletion of nucleotides Single strand breaksSingle strand breaks Double strand breaksDouble strand breaks Crosslinking—covalent linkage Crosslinking—covalent linkage

between basesbetween bases

Spontaneous MutationsSpontaneous Mutations Arise without mutagenic agents. DNA Arise without mutagenic agents. DNA

pol has proofreading function, can pol has proofreading function, can remove mismatched baseremove mismatched base

Even if DNA pol misses a mismatch Even if DNA pol misses a mismatch other systems can recognize and other systems can recognize and repair it.repair it.

Recognition?Recognition? Hemimethylation-allows enzymes to Hemimethylation-allows enzymes to

distinguish between parent and daughter distinguish between parent and daughter strands.strands.

Spontaneous MutationsSpontaneous Mutations Tautomeric shifts during replicationTautomeric shifts during replication.. Depurination—if a purine base is lost Depurination—if a purine base is lost

from C-1 of deoxyribose, will get from C-1 of deoxyribose, will get apurinic site.apurinic site. Odds of misincorporation on the Odds of misincorporation on the

daughter strand=75%daughter strand=75% Enzymes specific for this type of Enzymes specific for this type of

mutation have evolvedmutation have evolved Deamination. Deamination.

CCUU AAHypoxanthineHypoxanthine

Altered H-bonding}

Tautomers and MutationTautomers and Mutation

Rare imino forms of adenine and cytosineRare enol forms of thymine and guanine

Normal base Normal base pairingpairing

Back

Deamination of C and ADeamination of C and A CCUU

3 H-3 H-bonds bonds w/Gw/G2 2 H-bonds H-bonds w/Aw/A

AAHypo-Hypo-xanthinexanthine 2 H-2 H-

bonds bonds w/Gw/G3 3 H-bonds H-bonds w/Cw/C

Removing and Replacing Removing and Replacing UracilUracil

Uracil automatically removed from DNA Uracil automatically removed from DNA by uracil N-glycosylaseby uracil N-glycosylase

AP Endonuclease cuts 5’ to AP Endonuclease cuts 5’ to apurinic siteapurinic site

Sugar phosphate removed Sugar phosphate removed by phosphodiesterase by phosphodiesterase

DNA pol I adds correct baseDNA pol I adds correct base

Ligase sealsLigase seals Base Excision Repair (BER)Base Excision Repair (BER)

Base Excision Repair (BER) Base Excision Repair (BER)

P

A

P

G

P

G

P

C

P

T

P P

C

P

G

P

A

P

G

P

G

P

C

P

T

P

U

P

C

P

G

P

A

P

G

P

G

P

C

P

T

P

G

P

A

P

G

P

G

P

C

P

T

P

C

P

C

P

G

Uracil DNA glycosylase

AP endonuclease

DNA polymerase I

DNA ligase

5 Methyl Cytosine 5 Methyl Cytosine DeaminationDeamination

Easily Easily recognized recognized and and correctedcorrected

What about What about 5-methyl 5-methyl cytosine?cytosine?

Is there a Is there a problem?problem?

Always Always remove T remove T from a GT from a GT pairpair ?

Deamination of Cytosine and Deamination of Cytosine and 5-methylcytosine5-methylcytosine

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Induced MutationsInduced Mutations Caused by exposure to a mutagenCaused by exposure to a mutagen CausesCauses

Exposure to base analogsExposure to base analogs Chemical mutagensChemical mutagens Intercalating agentsIntercalating agents Uv- radiationUv- radiation Transposable elementsTransposable elements Mutator genesMutator genes

Exposure to Bases AnalogsExposure to Bases Analogs Base analogs—Base analogs—

substances that substances that are similar to and are similar to and can substitute for can substitute for standard basesstandard bases

Examples—AZT, 5-Examples—AZT, 5-bromouracil (5-BU) bromouracil (5-BU) and 2-aminopurine and 2-aminopurine (2-AP)(2-AP)

5 Bromouracil5 Bromouracil The Problem: 5 The Problem: 5

bromouracil bromouracil assumes the enol assumes the enol form at a much form at a much higher frequency higher frequency than Tthan T

if it replaces T, if it replaces T, will probably get a will probably get a mutation due to mutation due to tautomerization tautomerization during replicationduring replication

Result: AResult: A·T ·T G·CG·C

A·TA·TReplication in presence of BrU

A·BrUA·BrU

Tautomeric shift

A·BrUA·BrU**

Replication

A·T + A·T + GG·BrU·BrU**

Replication

A·T + AA·T + A·T·T + G + G·BrU·BrU** + GC + GC

THE PROCESS