DNA damage and replication stress

pathways of DNA repair

single stranded - BER, NER
double stranded - HR, NHEJ

base damage - 8oxoG

guanine - 8oxoG which can rotate causing recognition as a T, base pairs with A upon replication
GC to TA transversion

removal of 8oxoG

MTH1 removes from nt pool
OGG1 excises from a GC base pair
MUTYH excises from GA base pair

MUTYH mutations

MUTYH associated polyposis and CRC

base damage - oxidation

oxidation of cytosine to uracil
CG to TA transition

steps of BER

1. base flipping and removal by DNA glycosylases leaving abasic site

2. APEI cuts DNA backbone

3. SHORT PATCH = ribose phosphate portion of nucleotide removed, missing nt replaced by DNA polB, nick sealed by ligase III

4. LONG PATCH = DNA pol delta synthesises over abasic site, displaced flap of old strand cleaved by FEN1, nick sealed by ligase I

causes of nucleotide damage

DNA lesions caused by UV light
intra-strand cross links causes kinks in helix

steps of NER - damage detection

global genomic damage detection = RAD23B and XPC move along chromosome constantly scanning

transcription coupled damage detection = CSA and CSB dimer associates with RNAPII

steps of NER

1. damage detection

2. TFIIH unwinds 30 nts around damaged site

3. XPF/ERC1 and XPG endonucleases incise DNA round damage

4. gap filled by DNA synthesis and ligation

NHEJ or HR

NHEJ dominant in G1
HR restricted to S phase
HR dominant in lower eukaryotes
resection leads to HR

steps of NHEJ

1. ring like Ku protein binds ends

2. recruits DNA-PK to keep ends together

3. nuclease artemis cleans up ends to find microhomology at break

4. DNA ligase IV and XRCC4 and XLF ligates edited ends

NHEJ functions

required for VDJ recombination
defects lead to SCID

steps of HR

1. short range 5’ resection - CltP, MRN and MreII

2. OR long range 5’ resection - EXO1 may take over

3. RPA binds 3’ overhangs

4. BRCA2 facilitates Rad51 filament formation

5. homology search and strand invasion - sister chromatid D loop provides a primer

6. replication machinery fills gap

origins of DNA replication errors

defective MMR
TLS
STR replication

MMR

1. recognition by MSH2-MSH6

2. recruits MLH1

3. PCNA and RPA direct repair machinery

4. EXO1 removes mismatched base

5. pol delta uses parental strand as template to fill gap

detection of new strand - bacteria

hemi methylated strand

detection of new strand - eukaryotes

presence of nicks in newly synthesised strand

origin of nicks in newly synthesised strand

erroneous removal of NTPs
5’ end of okazaki fragment mismatch
endonuclease activity of MutL

TLS

DNA damage tolerance mechanisms that allows bypass of DNA lesions
DNAP will stall at lesions and TLS replicates these sites
TLS polymerases have no proof reading activity

TLS polymerases

Y family - PAD domain stabilises on DNA
pol zeta

role of TLS in SHM

AID deamidates C to U
U can be excised to create abasic site which is replicated by TLS - mutation
replication of abasic site stimulates recruitment of pol eta - further mutation

replication of STRs

leads to expansion - nascent DNA may dissociate and reassociate or form loops

origins of DNA replication stress

DNA damage
difficult to replicate DNA
secondary structures
transcription-replication conflicts
oncogene activation
chemotherapy

consequences of DNA replication stress

activation of ATR-CHK1 checkpoint
DSBs - rearrangements
aneuploidy
micronuclei
Oncogene induced RS

ATR-CHK1 checkpoint

1. lesion causes pol uncoupling from helicase and accumulation of ssDNA

2. RPA binds

3. ATR and CHK1 recruited

4. CHK1 inhibits S CDK, stabilises fork and suppresses origin firing

examples of oncogene induced RS

cyclin E causes excessive CDK2 activity
myc upregulates replication factors
ras causes increased mitogenic signalling

oncogene induced RS

excess origin firing leads to exhaustion of resources and RS - activation of checkpoints
cancer cells become dependent on p53 checkpoint to manage RS - selection pressure to remove p53 and prevent apoptosis

synthetic lethality - PARPis

inhibition of SSBR - reliance on DSBR
traps PARP on DNA - unable to replicate