DNA Repair Flashcards
sources of mutations (3)
- replication errors (not corrected by proofreading)
- spontaneous changes in DNA
- external factors (Radiation, temp, mutagens)
depurination meaning
remove base
deamination meaning
change base
transition mutation, base substitution, point mutation, transversion mutation
purine/pyrimidine -> purine/pyrimidine = transition mutation, base substitution, point mutation
purine -> pyrimidine = transversion mutation
lots of replication errors are corrected immediately by ___ and ___ ___
DNAP
mismatch repair
how many mistakes per cell division?
3 mistakes
spontaneous changes - deamination types
- cytosine -> uracil
- adenine -> hypoxanthine
- 5-methylcytosine -> thymine
spontaneous changes - depurination (A or G)
spontaneous hydrolysis of N-glycosyl linkage -> remove base
- can replace missing base or remove entire base pair:
- possible frame shift if unpaired base also deleted
external factors for mutation (list)
alkylating chemicals: e.g., nitrosamines, N-methyl-N-nitro-N-nitrosoguanidine
- alkyl groups (mostly methyls) added to DNA
reactive oxygen species (H2O2, O2-, OH radical) generated by ionizing radiation and chemical agents
- oxoG adduct - marker for oxidative stress
- highly mutagenic
base analogs: e.g., 5-bromouracil = thymine analogue, misspairs with guanine
external factors: what do bases misspair with?
alkylation:
- guanine -> O6methylguanine misspairs with T
oxidation
- guanine -> oxoG misspairs with A
base analogs
- thymine analogue = 5-bromouracil misspairs with G
what does nonionizing radiation result in?
(UV light) 260nm
=> thymine/thymidine dimer
what does ionizing radiation lead to?
(x rays, gamma rays)
double-strand breaks
- a lot of bases lost
what do intercalating agents lead to?
e.g., acridine, ethidium bromide
- flat, polycyclic molecules that insert b/w stacked DNA based
- can cause deletion or addition
thymine dimers (UV) details
- cyclobutane ring joins adjacent thymine bases
- distorts backbone (bulge), prevent proper base pairing
2 types of DNA repair enzyme systems
- constitutive
- damage-inducible
what repair methods repair replication errors (2)
proofreading of replisome:
- 3’-5’ exonuclease activity of pol
mismatch repair
- removes errors missed from proofreading during replication
- must be rapid (before next replication)
- distortion of DNA backbone -> detect mismatch
mismatch repair system - mut genes in E.coli (4)
MutS
- scans DNA for distortions
- binds to mismatch
MutL
- recruited by MutS
MutS translocates along DNA until GATC seq, NEED ATP, makes DNA loop
MutSL activates MutH (endonuclease)
- recognizes GATC, binds MutSL
MutH nicks UNmethylated DNA strand near GATC
mismatch repair system in E. coli cont’d
(after MutH nicks unmethylated DNA strand near GATC)
strand degraded from GATC to mismatch
- 5’-3’ direction (RecJ or exonuclease III) or
- 3’-5’ direction (exonuclease I)
- helicase helps
new DNA strand made
- DNA pol III
- DNA ligase
mismatch removed & corrected
what enzyme methylates the adenine of all GATC?
Dam Methylase
how does repair system which mismatched nucleotide should be replaced?
Dam Methylase hasn’t yet methylated the adenine of GATC site of new strand;
still HEMImethylated
- MutH nicks unmethylated strand!
higher euk possess Mut protein homologues
- MSH (MutS homolog: MutS alpha) and MLH/PMS (MutL homolog: MutL alpha) recognize and repair
5’ or 3’ excision following recognition of distortion
- MSH/EXO1 removes 5’ error
- MLH/PMS removes 3’ error by interacting with PCNA (endo)
- resulting gaps filled like in lagging strand (DNA pol delat)
direct reversal: repair of thymine dimers (what process and protein)
- through photoreactivation by Photolyase
- photoreactivation = light-dependent activity that breaks covalent bonds between thymines
- excision repair can also repair thymine dimers
2 types of excision repair
base excision repair
- damaged base removed from backbone
- repair: DNA pol, DNA ligase restore DNA
- lesion-specific: specific DNA glycosylases
nucleotide excision repair
- NOT lesion-specific, recognizes distortion, massive dmg/lesions
- cleavage on both sides of damage, removal and replacement of one strand
- DNA pol and ligase finish
base excision repair proteins/enzymes + pathways
2 pathways: pol beta-mediated (short patch) or pol delta/epsilon-mediated (long patch)
- DNA glycosylase recognizes, removes base by hydrolyzing glycosidic bond
- apurinic/apyrimidic endonuclease (APE1) removes abasic sugar
- PCNA sets DNA pol (beta or delta/epsilon)to fill gap
- FEN-1: removal of displaced strand
- DNA ligase 1 or 3
what do DNA glycosylases have the ability to do?
they are lesion-specific and diffuse along minor groove to detect the specific lesion
- have ability to flip out damaged base
what enzymes can flip bases out of DNA helix (3)
- glycosylases
- photolyases
- methylases
nucleotide excision repair proteins (uvr system in E. coli)
uvr system recognizes DNA distortion (thymine dimer)
- UvrA, B, C involved in recognition, incision, excision (repair endonuclease)
- UvrAB complex: scans DNA
- UvrA: detects distortion (not base cahnge)
- UvrB: unwinds DNA -> ss bubble around lesion; bubble recruits UvrC, UvrA dissociates (NEEDS ATP)
- UvrBC complex make 2 incisions on both sides of dmg (NEEDS ATP)
- UvrD (helicase binds and unwinds DNA)
- DNA pol I replaces damage…
“UvrBC complex make 2 incisions on both sides of dmg” how many nucleotides in each direction?
7 nts to 5’ side of dmg
3-4 nts to 3’ side of dmg
recombination repair
- no undamaged template is available (ex. double breaks, DNA nick during rep)
- filling gap by retrieving a corresponding single strand from another (homologous) duplex
2 types of error-prone repairs
- nonhomologous end joining (NHEJ)
- SOS translesion repair
nonhomologous end joing (NHEJ)
- no undamaged template avail
- two ends of broken DNA are ligated together:
- Ku70/Ku80 dimer recognizes broken ends, holds them together
- DNA-dep protein kinase (DNA-PK): brings and phosphorylates Artemis protein
- Artemis (exo + endo) trims overhands, cleaves hairpins
- DNA pol fills, ligase…
SOS translesion repair
- highly error-prone
- result of SOS response
- polymerases add nucleotides randomly without proper pairing
NHEJ vs Recombination Repair
- they both repair ds breaks
- difference: accuracy of repair
NHEJ (non-homologous, more error-prone)
recombination repair (homologous, error-free)
