10. DNA Repair

DNA structure great for repair

if one strand is damaged, the complementary strand serves as template for reapir

lots of replication errors are corrected immediately by

DNAP and mismatch reapir

frequency of mutations in male sperm is

about 10-50 fold higher

deamination of cytosine

changes C:G to U:A

deamination of adenine

adenine → hypoxanthine (which pairs with c, not T)

deamination of 5-methylcytosine

changes Cm:G to T:A

Depurination (A or G)

spontaneous hydrolysis of N-glycosyl linkage → abasic (apurinic) site in DNA

• possible Frame Shift in de-purinated strand after replication

alklylation

guanine → O6 methylguanine (miss-pairs with T)

Oxidation

guanine → 8-oxoguanine (miss-pairs with A via non WC base pairing)

Base analogues

5-bromouracil (miss pairs with G)

nonionizing radiation (UV light(

thymine dimers

ioinizing radiation (x rays, gamma rays)

double strand breaks

intercalating agents: acridine, ethidium bromide

flat, polycylic molecules that insert between stacked DNA bases

may cause deletion or addition of one-few base pairs whne polymerase subsequently skips or adds one or more nucleotides

thymine dimers (UV irradiation)

• cyclobutane ring joins adjacent thymine bases

• distorts backbone, prevents proper base pairing

proofreading of replisome

• 3’ - 5’ exonuclease activity of polymerase

• removes wrongly incorporated bases

• improves fidelity but mismatches still occur

mismatch repair

• removes errors that escape proofreading during replication

• must be rapid

• must find and correctly replace mismatch

• distortion of DNA back bone enables detection of mismatch

MutS

scans DNA for distortion & binds to mismatch

MutL

recruited by MutS-mismatch-DNA complex

MutS translocates along DNA until a GATC sequence is reached

• requires ATP

• bidning of MutS at the 2 locations creates loop in DNA

• MutSL activates MutH (endonuclease)

• MutH nicks unmethylated strand of DNA

how does repair system know which mismatched nucleotide should be replaced

the dam methylase will methylate the adenine of all GATC sites

when a mismatch occurs, MutH recognizes the other unmethylated strand and introduces a nick in it

MutSL system is often defective in

human cancers

repair of thymine dimers

direct reversal reapir is through phototoreactivation done by photolyase

base excision repair

• damaged base removed from backbone

• repair: DNA polymerase, DNA ligase restore DNA

• lesion-specific: specific DNA glycosylases

nucleotide excision reapir

• not lesion specific, recognizes distortion

• cleavage on both sides of damage, removal and replacement of one strand

• DNA polymerase and DNA ligase complete repair

4 enzymes involved in base excision repair

1. glycosylase recognizes, removes base by hydolyzing glycosidic bond

2. apurininic/apyrimidic endonuclease which removes abasic sugar

3. PCNA* (sliding clamp) positions DNA pol B to fill in the gap

4. DNA ligase seals nick

DNA glycosylases are lesion specific

diffuse along minor groove to detect specific lesion, have ability to flip out damaged base

nucleotide excision reapir steps

1. UvrAB complex scans DNA

2. UvrA detects distortion (not base change)

3. UvrB melts DNA → ss bubble around lesion bubble “recruits” UvrC: uvrA dissociates (ATP)

4. UvrC joins UvrB → UvrBC makes 2 incisions

5. UvrD (helicase) binds and unwinds DNA;

6. DNA Pol I replaced damaged strand

if nucleotide excision repair in humans on actively transcribed strand

transcription-coupled nucleotide excision repair pathway - rapid repair of transcriptionally active DNA

recombination repair activated by events which involve both strands of dsDNA

1. during replication - when replication fork encounters lesion in DNA that has not been repaired

2. if damage occurs at sample place in both strands of DNA - no undamaged complementary strand (template) us available

recombination pair happens in necessary for

double strand break (DSB) repair pathways; damage tolerance mechanisms; post replication repair

recombination repair mode of filling a gap in one strand fo duplex DNA by

retrieving a corresponding single strand from another (homologous) duplex

possible cause of damage in both strands

original dsDNA with dimer (damaged blue strand) was not fixed before replication

trigger for non-homologous end joining (NHEJ)

both DNA strands break at the same location and no intact complementary template exists

work as a fail-safe system

drawback for NHEJ

error-prone - can delete or insert nucleotides, altering the DNA sequence

trigger for SOS Translesion repair

severe DNA damage that blocks replication fork progression

SOS translesion error-prone because

polymerases insert nucleotides without correct base pairing