Deamination of Cytosine and 5-methylcytosine

1. Deamination of Cytosine and 5-methylcytosine -------------------------------------------------------------------------------

2. Chemical Mutagens

3. Intercalating Agents • EX, Ethidium bromide, acridine orange • Can induce frameshift mutations

4. Uv Induced Dimers • Thymine dimers and T-C dimers • Replication problems • Interferes with the ability of the T’s to base pair to the opposite strand, and blocks DNA replication

5. Other Mutagens • Transposable elements—”jumping genes”. • Major frameshift mutations • Factors in evolution • Mutator genes—mutations increase mutation rate. Four potent mutator genes • Mutant DNA pol III 3’5’ exonuclease activity • Mutant methylation enzymes(ex dam) • Mutant enzymes in excision repair system • Mutant enzymes in SOS system

6. Reversions • Mutations in an mutant can restore the wild type function (reversion, back mutation, or reverse mutation) • Spontaneous or induced • If mutation occurs at the site of the original=True reversion • Wild type restored by mutation at another site= second site mutation • Second site in same gene= intragenic suppression • Second site in another gene=intergenic suppression

7. Intragenic Revertant • Types • Same site reversion • Second site revertant • NOTE: shape of R-groups can also be a factor. EX

8. Reversion of Frameshift Mutations • For reversion to be successful • Reversion must be near original site to reduce # of aa altered • Section of polypeptide must be able to withstand alteration without eliminating function

9. Intergenic Suppression • Refers to a chnge in another gene which suppresses or eliminates the mutant phenotype. EX • Multisubunit proteins—Mutation in one subunit may be masked by mutation in another subunit (ex. restoring hydrophobic patches) • Suppression via suppressor tRNAs

10. Suppressor tRNA • Nonsense mutation- aa codon  “Stop” • Ex. AAGUAG • Some bacteria can “read through” these mutations (though protein function may be altered). HOW? • Mutant tRNA that has an anticodon that recognizes “Stop” as a reading codon. • aa encoded depends on which tRNA is mutated • Not every suppressor restores normal function

11. Suppressor Mutants (cont’d) • EX. UUG (Leu)UAG (Stop) (AUC anticodon) • A mutation in a tRNA resulting in “AUC” allows that tRNA to recognize “Stop”. • Can get suppression or partial suppression • NOTE: must be 2 copies of tRNA mutated. Why? • In any cell containing mutator, must also be a wild type • Suppressors allow survival, even if sub-optimal

12. Termination of Translation in Suppressor Strains • Problem: Must be a means of terminating translation. • HOW? • Release factors still present, will compete for the “Stop “ site • Many genes are double-terminated • EX. UAG-UAA

13. O6-methylguanine methyl transferase Fig. 20.40 Repair by Direct Reversal Photolyase Fig. 20.39

14. Most common repair mechanism EX. Uvr system NOTE: preferentially repairs dimers in essential regions of genome UvRA recognizes damage and binds w/UvrB UvRA released, UvrC binds UvrC nicks on both sides of damageUvrD unwinds region Damaged strand released DAN pol I Ligase Excision Repair

15. Recombinational Repair • Sister Strand Exchange

16. Recombinational Repair, aka, Sister Strand Exchange

17. SOS Response • Is an inducible system of last resort • Also called error prone replication because it inactivates the proofreading function of DNA pol III. • Turned on only when DNA damage is extreme • Main players: recA and lexA and a battery of inducible enzymes

18. SOS Response