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BIOL 308 Molecular Biology > DNA Repair > Flashcards

DNA Repair Flashcards

1
Q

sources of mutations (3)

A
  • replication errors (not corrected by proofreading)
  • spontaneous changes in DNA
  • external factors (Radiation, temp, mutagens)
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2
Q

depurination meaning

A

remove base

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3
Q

deamination meaning

A

change base

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4
Q

transition mutation, base substitution, point mutation, transversion mutation

A

purine/pyrimidine -> purine/pyrimidine = transition mutation, base substitution, point mutation

purine -> pyrimidine = transversion mutation

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5
Q

lots of replication errors are corrected immediately by ___ and ___ ___

A

DNAP
mismatch repair

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6
Q

how many mistakes per cell division?

A

3 mistakes

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7
Q

spontaneous changes - deamination types

A
  • cytosine -> uracil
  • adenine -> hypoxanthine
  • 5-methylcytosine -> thymine
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8
Q

spontaneous changes - depurination (A or G)

A

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

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9
Q

external factors for mutation (list)

A

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

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10
Q

external factors: what do bases misspair with?

A

alkylation:
- guanine -> O6methylguanine misspairs with T

oxidation
- guanine -> oxoG misspairs with A

base analogs
- thymine analogue = 5-bromouracil misspairs with G

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11
Q

what does nonionizing radiation result in?

A

(UV light) 260nm
=> thymine/thymidine dimer

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12
Q

what does ionizing radiation lead to?

A

(x rays, gamma rays)
double-strand breaks
- a lot of bases lost

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13
Q

what do intercalating agents lead to?

A

e.g., acridine, ethidium bromide
- flat, polycyclic molecules that insert b/w stacked DNA based
- can cause deletion or addition

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14
Q

thymine dimers (UV) details

A
  • cyclobutane ring joins adjacent thymine bases
  • distorts backbone (bulge), prevent proper base pairing
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15
Q

2 types of DNA repair enzyme systems

A
  • constitutive
  • damage-inducible
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16
Q

what repair methods repair replication errors (2)

A

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

17
Q

mismatch repair system - mut genes in E.coli (4)

A

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

18
Q

mismatch repair system in E. coli cont’d
(after MutH nicks unmethylated DNA strand near GATC)

A

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

19
Q

what enzyme methylates the adenine of all GATC?

A

Dam Methylase

20
Q

how does repair system which mismatched nucleotide should be replaced?

A

Dam Methylase hasn’t yet methylated the adenine of GATC site of new strand;
still HEMImethylated
- MutH nicks unmethylated strand!

21
Q

higher euk possess Mut protein homologues

A
  • 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)
22
Q

direct reversal: repair of thymine dimers (what process and protein)

A
  • through photoreactivation by Photolyase
  • photoreactivation = light-dependent activity that breaks covalent bonds between thymines
  • excision repair can also repair thymine dimers
23
Q

2 types of excision repair

A

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

24
Q

base excision repair proteins/enzymes + pathways

A

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
25
Q

what do DNA glycosylases have the ability to do?

A

they are lesion-specific and diffuse along minor groove to detect the specific lesion
- have ability to flip out damaged base

26
Q

what enzymes can flip bases out of DNA helix (3)

A
  • glycosylases
  • photolyases
  • methylases
27
Q

nucleotide excision repair proteins (uvr system in E. coli)

A

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…

28
Q

“UvrBC complex make 2 incisions on both sides of dmg” how many nucleotides in each direction?

A

7 nts to 5’ side of dmg
3-4 nts to 3’ side of dmg

29
Q

recombination repair

A
  • no undamaged template is available (ex. double breaks, DNA nick during rep)
  • filling gap by retrieving a corresponding single strand from another (homologous) duplex
30
Q

2 types of error-prone repairs

A
  • nonhomologous end joining (NHEJ)
  • SOS translesion repair
31
Q

nonhomologous end joing (NHEJ)

A
  • 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…
32
Q

SOS translesion repair

A
  • highly error-prone
  • result of SOS response
  • polymerases add nucleotides randomly without proper pairing
33
Q

NHEJ vs Recombination Repair

A
  • they both repair ds breaks
  • difference: accuracy of repair

NHEJ (non-homologous, more error-prone)
recombination repair (homologous, error-free)

BIOL 308 Molecular Biology (14 decks)

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