Lecture 6 handout.pdf

Micro/Genome 411 Lecture 6 MUTATION & DNA REPAIR I. Methyl-directed mismatch repair A. DNA Adenine Methylase ("DAM methylase") B. Mechanism of mismatch repair (MutSHL) C. Genetic dissection of the system 1. Mutational spectra of mismatch mutants 2. Mutation frequencies of mismatch mutants II. Chemical damage to DNA bases A. Cytosine deamination 1. Uracil-N-glycosylase (UNG) 2. DNA Cytosine methylase (DCM) and mutational "hotspots" 3. "Very short patch" (vsp) repair B. Depurination C. Oxidative damage 1. Mutational spectrum 2. Repair of oxidative damage (MutT, MutM, MutY) 3. Phenotype of MutT/MutY double mutants III. Spontaneous frameshift mutations A. Frameshift "hotspots" B. Replication slippage events ? G A T C ? ? C C A G ? G G T C ? ? C C A G ? G A T C ? C T A G Replication (w/out repair) ? G A T C ? ? C T A G ? G G T C ? ? C C A G Repair (w/out selection) Lecture 6: Mutation & DNA Repair Micro/Genome 411 ? G A* T C ? ? C T A* G (Fully methylated DNA) (Hemi-methylated DNA) (Fully methylated DNA) ? G A* T C ? C T A* G ? G A* T C ? C T A* G ? G A* T C ? C T A G ? G A T C ? C T A* G Mutation frequencies of mutators defective for the same repair system: StrainMutation frequency Wild-type10 -10 MutS ? (Mismatch repair ? ) -9 MutH) 10 -9 MutS ? MutH ? (Double mutant) Mutation frequencies of mutators defective for different (redundant) repair systems acting on the same type of damage: StrainMutation frequency Wild-type10 -10 MutS ? (Mismatch repair ? ) -9 Proofreading ? (Leaky)10 -9 MutS ? Proofreading ? (Double) Mutation frequencies of mutators defective for different repair systems acting on different types of damage: StrainMutation frequency Wild-type10 -10 MutS ? (Mismatch repair ? ) -9 UvrA (Excision repair) 10 -9 MutS ? UvrA ? (Double mutant) ? G A T C ? ? C T A G ? G A T U ? ? C T A G Deamination ? G A T C ? ? C T A G ? G A T U ? ? C T A A Replication ? G A T U ? ? C T A A ? G A T T ? ? C T A A Replication G:C ? A:T transition "hotspot" sequence ? C C*A G G ? ? G G T C*C Lecture 6: Mutation & DNA Repair Micro/Genome 411 Depurination: The spontaneous cleavage of purines from dsDNA. O 2 O 2 ? H 2 O OH ? H 2 O e ? e ? e ? e ? Reduction of molecular oxygen during respiration Guanine8-oxo-Guanine (GO) G:C ? T:A transversions as a result of oxidative damage: ? A G C T ? T C G A A:T ? C:G transversions as a result of oxidative damage: ? A G C T ? ? T C G A Lecture 6: Mutation & DNA Repair Micro/Genome 411 lacI frameshift "hotspot" sequence motif: CTGG CTGG CTGG (wild-type) Frameshift mutants are typically "+1" or "-1" repeat: CTGG CTGG CTGG CTGG (frameshft) CTGG CTGG (frameshft) Replication "slippage" involving direct tandem repeats: + Kendall Gray Microsoft Word - Lecture 6 handout.doc