MOLECULAR BASIS OF MUTATION

KISHOR SAWAIKARR A C WASHIM (MS) INDIA

Substances and preparations which, if they are inhaled or ingested or if they penetrate the skin;

may induce cancer or increase its incidence and can affect any cells or tissues = Carcinogens

may induce hereditary genetic defects or increase their incidence and effect the germ cells (gonads) = Mutagens

may induce non-hereditary congenital malformations or increase their incidence and effect the growing foetus =Teratogens

DNA: The Hereditary Material Composition:DNA is a Nucleic Acid composed of a

long polymer of nucleotidesEach nucleotide is made up of: a- a sugar molecule (deoxyribose) b- a phosphate molecule c- a nitrogenous base

The nitrogenous bases are of two types:

a- Purines (Adenine and Guanine)

b- Pyrimidine (Cytosine and Thymine)

Watson & Crick double helix model (Nobel Prize winners)

Features:1- Two antiparallel strands2- One strand is 5´-3´ direction, the

other runs in the opposite 3´-5´ direction

3- Guanine on one strand always pairs with cytosine on the other strand and adenine always pairs with thymine

The induction of mutations is accomplished by treating cells

with mutagens. The mutagens most commonly used are high-energy radiation such as X-rays, UV rays or specific chemicals; examples of these mutagens and their efficacy are :

The intercalating agents are another important class of DNA modifiers. This group of compounds includes proflavin, acridine orange, and a class of chemicals termed ICR compounds , base analoges such as 5-bromouracil, Amino purine, Ethyl methane sulfonate, Nitrous acid, Methyl methane sulfonate, Nitrosoguanidine, Acridine mustard.

Molecular Basis of Gene Mutation Mutations are changes in the DNA sequence of genes.

Point mutations typically refer to alterations of single base pairs of DNA or of a small number of adjacent base pairs.           Mutations in DNA cause substitutions in protein  

New mutations are categorized as induced or spontaneous .Induced mutations are defined as those that arise after

purposeful treatment with mutagens, environmental agents that are known to increase the rate of mutations

. Spontaneous mutations are those that arise in the absence of known mutagen treatment. They account for the "background rate" of mutation and are presumably the ultimate source of natural genetic variation that is seen in populations.

The frequency at which spontaneous mutations occur is low, generally in the range of one cell in 105 to 108

Mutation can be classified as: a- germline - affecting tissues that produces eggs & sperm  ( heritable meiotically between generations)

b- somatic  - affecting other body tissues (heritable mitotically, e.g. cancer)

Molecular mechanisms of DNA mutagenesis:  Nucleotide interchanges are of two types:      1-   transition  is the replacement of a base by the other base of the same chemical category (purine replaced by purine: either A to G or G to A; pyrimidine replaced by pyrimidine: either C to T or T to C).

     2-   transversion is the replacement of a base of one chemical category by a base of the other (pyrimidine replaced by purine: C to A, C to G, T to A, T to G; purine replaced by pyrimidine: A to C, A to T, G to C, G to T).  

        Most mutations are transitions: interchanges of bases of same shape

At the DNA level, there are two main types of point mutational changes:

base substitutions and base additions or deletions.

Genetic Consequences of Mutation :

    Single-base mutations - interchanges of one DNA base type for another recognized as SNPs (single nucleotide polymorphisms)

    Consequences of exon mutations depend on position in triplet (examples)            

3rd position                  typically a silent mutation - if position "wobbles", no change to amino acid                  sometimes a missense mutation - results in different amino acid

           2nd position - always a missense mutation           

1st position - almost always a missense replacement                                       [Leu codons are major exception]

           Stop codon mutations may occur at any position:

                nonsense (termination) mutation terminates polypeptide prematurely                   

Missense mutations in DNA cause replacement substitutions in protein          Consequences depend on position of substitution in polypeptide

    none:  substitution not in active site or binding site

    minor: substitution of same type (synonymous substitution) allozymes are proteins with minor changes in electric charge

    major: substitution affects 20, 30, or 40 structure (nonsynonymous substitution)              Ex.: Sickle-cell hemoglobin (HbS)                        glu - val  in sixth residue of beta chain                         because GAG  GUG in sixth codon of beta-chain mRNA

                            

1- Silent substitutions: the mutation changes one codon for an amino acid into another codon for that same amino acid.

2- Missense mutations: the codon for one amino acid is replaced by a codon for another amino acid.

3- Nonsense mutations: the codon for one amino acid is replaced by a translation termination (stop) codon.

Insertion / Deletion (indel) mutations Gain or loss of one or more nucleotides produces:

        Frameshift mutations ( triplet reading frame offset )

1- Single & double nucleotide indel => all downstream amino acids change (e.g.)

nonsense mutation eventually (quickly) produced

2- Triplet indel - insertion / deletion of single amino acid produce milder consequences

3-Multiple triplets may produce major effects (e.g.)length mutations - larger indels (102~6 bps) may affect cytology of chromosomes ("Fragile X", )

Variable numbers of trinucleotide repeats occur in human genetic disorders         

  e.g.  Huntington's Disease              Gene has (CAG)n repeat near 5' end             # copies in affected individuals inversely correlated with age of onset             predicted mRNA & huntingtin protein with (gln)n repeat have been isolated             autosomal dominant inheritance: HD alleles 'dominate' standard alleles             phenotype is a consequence of presence of modified protein:                 

 e.g.  Fragile-X Syndrome [Martin-Bellyndrome] Most common form of inherited predisposition to mental retardation           (CGG)n repeat occurs upstream from 5' end of FMR-1 gene

# CGG repeats Geno / Phenotype

   6 ~ 54 standard

~ 55 ~ 200 carrier

> 200Fragile-X Syndrome

<>     X-linked inheritance

           Asymptomatic, hemizygous fathers pass "fragile X" chromosome to daughters

           Heterozygous daughters transmit to 1/2 of sons, who show syndrome                                                                         1/2 of daughers are carriers

                Syndrome becomes more pronounced in successive generations                 expansioin of repeat occurs in female germline

Mutations occur also in: 1- Regulatory and other noncoding sequences. Those

parts of a gene that are not protein coding contain a variety of crucial functional sites. At the DNA level, there are sites to which specific transcription-regulating proteins must bind.

2- At the RNA level, there are also important functional sequences such as the self-ligating sites for intron excision in eukaryote mRNAs.

It is important to realize that such regulatory

mutations will affect the amount of the protein product of a gene, but they will not alter the structure of the protein.

Alternatively, some mutations might completely inactivate function (such as polymerase binding or intron excision) and be lethal.

Mehanisms of Induced mutation Mutagens act through at least three different mechanisms. They can replace a

base in the DNA, alter a base so that it specifically mispairs with another base, or damage a base so that it can no longer pair with any base under normal conditions.

    1-alkylation - addition of alkyl group (methyl CH3- or ethyl CH3-CH2-))           ethyl methane sulfonate (EMS) is a common laboratory mutagen

       G - 6-ethyl G , pairs with T      2- deamination - conversion of amino into keto group 

nitrous acid (HNO2) is a common food additive / preservative

       C-  U by loss of NH2, pairs with A        A hypoxanthine by loss of NH2, pairs with C

 3- depurination - loss of purine (A or G) base in intact nucleotide produces an apurinic  site

replacement of base may produce transversion [a common form of damage in 'ancient' DNA] apyrimidinic sites are similar: loss of C or T

4- intercalation - base analogue wedged into DNA double helix [acridine dyes resemble 3-ring base pair ]  intercalated analogues read as 'extra' base:            DNAPol "stutters" => frameshift mutations                      (ethidium bromide is a fluorescent dye of DNA)

chemical modification of chemical modification of DNADNA changes base pairing changes base pairing

5-Tautomeric shifts All of the bases in DNA can exist in one of several forms, called

tautomers, which are isomers that differ in the positions of their atoms and in the bonds between the atoms.

The forms are in equilibrium. The keto form is normally found in DNA The enol forms of the bases are rare. The complementary base pairing of the enols is different from that of

the keto forms. For example, assume that a guanine in DNA changes into its enol

form at the moment at which it is copied in the course of replication, the enol form will bind to an incoming thymine. Hence we can represent the mutagenic process as follows, in which G* is the enol form of guanine:

6- Oxidatively damaged bases constitute another type of spontaneous lesion implicated in mutagenesis.

Active oxygen species, such as superoxide radicals (O2D), hydrogen peroxide (H2 O2) and hydroxyl radicals (OHD), are produced as by-products of normal aerobic metabolism.

These oxygen species can cause oxidative damage to DNA, as well as to precursors of DNA (such as GTP), resulting in mutation.

Genetic diseases are highly polymorphic with respect to their mutational basis

          PAH locus occurs near the end of short arm of Chromosome 12 (12p13.33)      14 exons produce a 2.4kb mRNA and a 452 amino acid polypeptide

      Of 66 alleles known to affect expression at the PAH locus 

29% produce non-PKU hyperphenylalanemia      71% produce PKU: of these,      14% are splice-site mutations      22% are deletion mutations (single base whole exon)      18% are "non-sense" mutations      55% are miss-sense mutations (39% of total)                       

Spontaneous mutations and human diseases.

DNA sequence analysis has revealed the mutational lesions responsible for a number of human hereditary diseases.

Many are of the expected simple base-substitution or deletion or addition type.

A number of these disorders are due to deletions or duplications of repeated sequences.

e.g. mitochondrial encephalomyopathies are a group of

disorders affecting the central nervous system or the muscles. They are characterized by dysfunction of mitochondrial oxidative phosphorylation and by changes in mitochondrial DNA structure.

These disorders have been shown to result from deletions of DNA sequences that lie between repeated sequences.

e.g. Fabry disease. This inborn error of metabolism results from mutations in the X-linked gene encoding the enzyme a-galactosidase Many of these mutations are gene rearrangements, resulting from either deletions or duplications between short direct repeats. All these deletions occur either by a slipped mispairing mechanism.

A common mechanism responsible for a number of genetic diseases is the expansion of a three-base-pair repeat, as in the Fragile X syndrome.

For this reason, they are termed trinucleotide repeat diseases. Fragile X syndrome is the most common form of inherited mental retardation, occurring in close to 1 of 1500 males and 1 of 2500 females. It is manifested cytologically by a fragile site in the X chromosome that results in breaks in vitro. Fragile X syndrome results from changes in the number of a (CGG )n repeat in the coding sequence of the FMR-1 gene.

The trinucleotide repeats result abnormal polypeptides containing large repeats of a single amino acid somehow contribute to the disease state.

The proposed mechanism for these repeats is a slipped

mispairing during DNA synthesis. However, the extraordinarily high frequency of

mutation at the trinucleotide repeats in fragile X syndrome suggests that in human cells, after a threshold level of about 50 repeats, the replication machinery cannot faithfully replicate the correct sequence and large variations in repeat numbers result.

X-linked spinal and bulbar muscular atrophy (known

as Kennedy disease ) results from the amplification of a three-base-pair repeat in this case, a repeat of CAG. Kennedy disease, which is characterized by progressive muscle weakness and atrophy, results from mutations in the gene that codes for the androgen receptor.

Normal persons have an average of 21 CAG repeats in this gene, whereas affected patients have repeats ranging from 40 to 52.

Myotonic dystrophy, the most common form of

adult muscular dystrophy, is yet another example of sequence expansion causing a human disease.

Susceptible families display an increase in severity of the disease in successive generations; this increased severity is caused by the progressive amplification of a CTG triplet at the 3 end of a transcript.

Normal people possess, on average, five copies of the CTG repeat; mildly affected people have approximately 50 copies; and severely affected people have more than 1000 repeats of the CTG triplet.

Additional examples of triplet expansion are still appearing for instance, Huntington disease, which has recently been added to the list.

Biological Repair Mechanisms

Living cells have evolved a series of enzymatic systems that repair DNA damage in a variety of ways.

The low spontaneous mutation rate is indicative of the efficiency of these repair systems.

Failure of these systems can lead to a higher mutation rate.

A number of human diseases can be attributed to defects in DNA repair, as we shall see later. Let's first examine some of the characterized repair pathways and then consider how the cell integrates these systems into an overall strategy for repair.

Repair defects and human diseases.

Several recessive human genetic diseases are known or suspected to be caused by defective genes in repair systems.

These defects often lead to an increased incidence of cancer because, as part of the general increased level of mutation, more mutations are produced in genes that can cause a cell to become cancerous.

One cancer-prone disease, xeroderma pigmentosum (XP), results from a defect in any one of eight genes involved in nucleotide excision repair. People suffering from this disorder are extremely prone to UV-induced skin cancer) as a result of exposure to sunlight) and have frequent neurological abnormalities.

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