Chapter 10

Gene Mutation:Origins and Repair Processes

Mutation is the process wherby genes change fromone allelic form to another.

Genetic analysis would not be possible withoutmutants.

Gene Gene mutations mutations at at the molecular levelthe molecular level

Triplets code for same amino acid: AGG ⇒ CGG (Arg)

Codon specifies a chemically similar amino acid

Codon specifies a chemically dissimilar amino acid

Forward mutations at protein level

Silent mutation

Synonymous missence mutation

Nonsynonymous missence mutation

Purin replaced by different purin, or pyrimidine replacedby a different pyrimidine: AT ⇒ GC; CG ⇒ TA

Purine replaced by a pyrimidine, or pyrimidine replacedby a purine: AT ⇒ CG; CG ⇒ GC

Forward mutations at DNA level

Transition

Transversion

ExamplesType of mutation

For example, compensation of a frameshift mutation bya second mutation

Intragenic suppressor mutation

forward reverseAAA(Lys) ⇒ GAA(Glu) ⇒ AAA(Lys)Wild-type Mutant Wild-type

forward reverseUCC(Ser) ⇒ UCG(Cys) ⇒ AGC(Ser)Wild-type Mutant Wild-type

forward reverseCGC(Arg) ⇒ CCC(Pro) ⇒ CAC(His)Wild-type Mutant Pseudo wild-type

Reverse mutations

Exact reversion

Equivalent reversion

Nonsense suppressors, missense suppressors,frameshift suppressors, physiological suppressors

Extragenic suppressor mutation

Any addition or deletion of base pairs that is not amultiple of 3.

Frameshift mutation

ExamplesType of mutation

Consequences of point mutations

Salvatoria Luria Salvatoria Luria and Max and Max Delbrück's fluctuation Delbrück's fluctuation testtestHypothesis: random mutation or directed physiological change (1943)

The frequency of mutations can be enhanced by mutagend

Mechanism of point-mutation induction

a) Base replacement by base analogs

All bases can exist in one of several forms, called tautomers, which areisomers that differ in the positions of their atoms and in the bondsbetween the atoms.

Mutation by tautomeric shifts

Alternative pairing possibilities for 5-bromouracil. 5-BU is an analog ofthymine that can be incorporated into DNA. 5-BU switches relativelyfrequently to the ionized form.

Mechanisms of mutation inductionb) Base alteration

Some mutagens alter a base, causing specific mispairing:

C

2

H

5

O

S

C

H

3

O

O

Ethyl methanesulfonate

Mechanisms of mutation inductionc) intercalating agents

Intercalating agents inhibit DNA synthesis and cause small deletions orinsertions

Mechanisms of mutation inductiond) Base damage

Many mutagens damage bases with the consequence that nospecific base pairing is possible.

Examples: UV light, Aflatoxin B1.

The SOS systemDNA polymerase stops at a noncoding lesion, generating ssDNA that attracts SsB and RecA whichforms filaments. These filaments stimulate the expression of UmuD. UmuD is cleaved by RecA toyield UmuD' and UmuC which permit the DNA polymerase to proceed with DNA synthesis.Mutations are caused, because the inserted bases have a high error frequency.

UV light generates pyrimidine photo dimers.

A cyclobutan and a6-4 photoproductcan be formed.These dimersinterfere with normalbase pairing.

Ames test

Cytochrom P450monooxygenases in the liverconvert certain metabolites byhydroxylation into mutagens.

Ames test showing themutagenicity of aflatoxin.

Salmonella strain TA100 is sensitive toreversion through base substitution.

Strains TA1538, TA1535 are sensitiveto reversion by frameshift mutation.

Binding of metabolically activated aflatocin

Spontaneous MutationSpontaneous lesions are caused bydeamination or depurination.DNA replication errors can also leadto mutations.

Deamination of 5-methlyctosin generates a "normal" base

Damage products formed attack by oxygen radicals

dR: deoxyribose

Biological repair mechanisms

1. Photolyase2. Excision repair3. Nucleotide exision repair4. Mismatch repair5. Post-replication repair

Photolyases catalyse the repairof pyrimidine dimers.

The enzyme uses energy fromvisible light to break the carbon-carbon bonds that join adjacentpyrimidine residues after UVirradiation.

The base excision repair pathway.

In this example, a uracil that was formed bydeamination of cytosin is removed from thesugar-phosphate backbone by DNAglycosylase. These enzymes create apurinic orapyrimidinic (AP) sites. AP endonucleaserecognizes these AP sites and cleaves theDNA strand. The remaining deoxyribosemolecule is removed by deoxyribosephosphodiesterase and the gap is filled.

Nucleotide excision repair (of apyrimidine dimer).

The DNA replication machinery is usedto remove distortions in the DNAdouble helix. Damaged DNA is firstrecognized by endonucleases andcleaved. The oligonucleotide is thenexised after helicase unwindes theDNA at this site.

Eukaryotic nucleotide excisionrepair in the context ofchromatin.

Highly conserved among eukaryotes.About 25 proteins are needed for theordered process of damagerecognition, excision of damagedDNA and repair synthesis. For repair,about 100bp of nucleosome free DNAare required. Nucleotide excisionrepair (NER) proteins are mobile. AfterDNA damage, NER proteins assembletransiently at the site of DNA lesion.One repair event needs ca. 4 minutes.After repair, the chromation assemblyfactor CAF-1 restores the chromatinstructure.

Xeroderma pigmentosum, a humandisorder, results from a defect in anyone of eight genes involved innucleotide excision repair.

Mismatch repair in E. coli.

Mismatch repair corrects errors madeduring DNA replication. A complex ofthree proteins (MutH, MutL, MutS)recognizes mismatched basesintroduced by DNA polymerase into thenewely synthesized, unmethylated DNAstrand. MutH cleaves the new strandopposite the methylated site and thedamaged DNA is then removed.

Post-replication repair.

Extensive DNA damage can be repairedby homologous recombination. Anunrepaired lesion can be bypassed byDNA polymerase. The resulting gap isthen repaired by recombination with theundamaged parental strand.

Mutational analysis

1. Somatic versus germinal mutation2. Mutant sectors3. Mutant phenotypes

Somatic versus germinal mutation

Mutant sectors

Summary