Mutagenesis in Growing Cells Double helical structure of DNA Watson and Crick (1953) Landmark achievement in Biology Impact:

Continuity of transmission from parent to progeny Storage of information in the form of base sequence Generation of mutation by uncorrected replication errors Other Sources of Spontaneous mutation: a) Exogenous DNA damage: Chemicals, UV etc., Error prone repair Mutations
b)

Transposition by mobile genetic elements Endogenous DNA damage: enzymatic, oxidative etc., cause mispairing Mutations -NH2 5- Me Cytosine (pairs with Guanine) Me C G G A 5-Me Uracil (Thymine) (pairs with Adenine) Me U (T) T

c)

Mutagenesis by replication errors A T A G C C

Nucleotide polymerisation rate: Approx. 5000 bases/sec Therefore a good degree of error is unavoidable. Most of the errors corrected by repair systems. Uncorrected ones lead to mutations. This idea was implicit in the double helical structure of DNA Evidence: Provided by Speyer (1965) Studied the reversion of T4 r II mutants to r+ in gene 43 mutant backgrounds Reversion RII r+ Cannot develop on Hosts lysogenic for Can develop on lysogens

Even 1r+ in a population of 108 109 r II can be detected. Very sensitive. Temporal cascade of events in phage infection. Infection Entry of phage DNA into host cells Immediate early gene expression (up to 2-3 min) Delayed early gene expression (up to 10' or so) phage DNA replication, arrest of host DNA replication, degradation of host DNA etc., Late gene expression Synthesis of viral structural components Virus assembly Lysis and releases of progeny virus.

In the case of T4 one of the products of early gene expression is a phage specific DNA polymerase (product of Gene 43) used to replicate T4 DNA Speyer constructed double mutants harbouring a given r II mutation and various gene 43 mutations r II 431, r II 432, r II 433 etc., Measured the r II r+ reversion frequency using each of the double mutants Three classes of gene 43 mutants I Had no effect on the event studied II Increased the reversion frequency Mutators (L 56, L 98) III Decreased the reversion frequency Anti Mutators (L 141,L 42) Replication A Accuracy of Base Selection (Fidelity): Commission of Errors B Correction of errors A B = Uncorrected replication errors Mutations In 1965 people were unaware of error correction Normal fidelity Normal error frequency Normal mutation frequency Decreased fidelity Increased error frequency Increased mutation frequency Mutators Increased fidelity Decreased error frequency decreased mutation frequency Anti mutators

Purified T4 DNA polymerase (Gene 43 encoded protein) A single polypeptide NTerminal domain C Terminal Domain Has 3 ' 5' Exonuclease activity a) Polymerase activity b) 5' 3 ' Exonuclease activity Mutators (L 56, L 58) Anti mutators (L 141, L 42 ) have lesions in this part have lesions in this part Low 3 5 Exo activity would allow more errors to escape correction Hence mutator activity It is believed that in anti mutator mutant errors are more accessible for correction than normal

Correction of replication errors in E.coli T4: Both DNA polymerase and 3 ' 5' Exonuclease activities reside in the same protein E.coli: DNA replication is a very complex process involving the concerted action of 10 proteins DnaE gene product: DNA polymerase III (polC) protein involved in dNTP polymerisation dnaQ (mut D) gene product: 3 ' 5' Exonuclease

dnaQ (mutD) mutants are very strong mutators

Wild Type ( Normal ) dnaQ49 dnaQ902 mutD5

Rif S Rif R frequency 1 1400 200 3500

Both mutator and anti mutator mutations in dnaE have been isolated. Simple explanation: Decreased fidelity in base selection: Mutator Increased fidelity in base selection: Anti mutator By and large true, but does not hold good for all mutations What if a replication error is not corrected then and there? A C Another process called Post-replication repair (also called Methyl Directed Mismatch Repair MMR) comes into play. Corrects mismatches after replication has proceeded beyond the mismatched base pair Problem: Progeny strand corrected with respect to parent correct repair: should occur

Parent strand corrected with respect to progeny strand wrong repair : should not occur

Parent Progeny

A C

A C G C Wrong Repair

A T Correct Repair

There should be a mechanism to distinguish between parent and progeny strands How is this achieved? In E.coli wherever the sequence GATC occurs the A is methylated (GA*TC) Methylation is a post-replicative event, done by an enzyme DNA-adenine methylase, the product of the dam gene. There is a time lag (window) between replication and methylation

Hemi methylated DNA Parent Progeny Distinction Possible MMR should occur

Fully methylated DNA Parent Progeny distinction not possible Repair during this state

during this state

is a chance event

dam o mutants: Mutators : MMR affected dam (under active ) mutants: Mutators : Even parent strand not optimally methylated.Impaired parent progeny distinction dam (over producer ) mutants : Mutators :Progeny strand methylated sooner than normal. Reduction in the Time window MMR Under the control of 3 Genes mut S mut L mut H Mismatch Repair in Salmonella Done by hex A and hex B gene products Recognition provided by gaps in the progeny strand (Okazaki fragments) Repair has to occur before the gaps are sealed Mutations in any Mutator effect