DNA damage response Flashcards
What type of things cause constant damage to our DNA?
- Genotoxic agents (endo/exogenous)
- Nucleotide misincorporation during DNA replication
- Intrinsic biochemical instability of DNA itself
What may failure to repair DNA lesions result in?
- Blockage of transcription/replication
- Mutagenesis
- Cellular cytotoxicity
What does DNA damage lead to the activation of?
Cell cycle checkpoints that pause the cell cycle to give time to repair the damage.
Failure in these checkpoints can lead to an accumulation of damage, which leads to mutations.
During replication, what does the error rate of DNA polymerase determine?
The spontaneous mutation rate in an organism.
Proofreading enzyme usually corrects most of these errors, however some mutations survive.
What are the main DNA repair mechanisms?
- Mismatch repair (MMR)
- Base excision repair (BER)
- Nucleotide excision repair (NER)
- Direct repair
DNA ds-breaks are repaired by:
- Homologous recombination (HR)
- Non-homologous end joining (NHEJ)
Provide an overview of mismatch repair (MMR)
Recognises erroneous insertions, deletions and misincorporation of bases that arise during DNA replication/recombination.
‘Mut’ proteins are the major active components of the mismatch repair system.
Mutations in human homologues of the Mut proteins affect genomic stability, which can result in MSI (frequently implicated in cancer).
E.g. MSH2/MSH6/MLH1 encode Mut proteins - mutations in these are commonly found in HNPCC/Lynch.
Provide an overview of base excision repair (BER)
Primarily for removing small, non-helix-distorting base lesions from the genome. Downstream steps of BER also used to repair single-strand breaks.
Involves DNA glycosylases (recognise/remove initial specific damaged/inappropriate bases, forming AP sites), plus AP endonucleases, DNA polymerase + ligase.
Deletion of BER genes increases mut rate in variety of organisms, which could contribute to development of cancer.
BER excision repair defects are not common in cancer but rare homozygous defects in MUTYH repair enzyme are associated with increased risk to colon cancer.
Provide an overview of nucleotide excision repair (BER)
Removes thymine dimers caused by UV radiation and large chemical adducts.
2 classes that differ in initial steps:
1. Global excision repair (repairs all DNA) - activated by binding of DDB and XPC to repair sites.
2. Transcription-coulped repair (only DNA undergoing transcription) - activated where RNA polymerase has stalled.
After the initial recognition steps, a cascade pathway is activated that results in unwinding of DNA helix, excision of damaged area and repair by low fidelity polyermases.
NER is defective in cancer prone syndromes such as Xeroderma Pigmentosum and Cockayne syndrome.
There are two alternatives for repair of ds-DNA breaks. Can you talk us through homologous recombination?
Involves BRCA1/2 - after DNA damage, BRCA1 is phosphorylated and relocalises along with RAD51 to the damaged regions activating homologous repair.
Uses sister chromatids during G2 phase of the cell cycle.
Requires a homologous sequence with extensive sequence identity to guide repair, plus Rad51 strand exchange protein to catalyse invasion of the homologous duplex sequence by the 3’ end of the ssDNA to replace the equivalent strand.
Other genes involved include NBS and BLM (which cause Nijmegen breakage/Bloom syndromes).
There are two alternatives for repair of ds-DNA breaks. Can you talk us through homologous recombination?
Ds-breaks directly repaired without the need for a homologous template.
Typically uses short homologous DNA sequences called microhomologies to guide repair, which are often present in single-stranded overhangs on the ends of double-strand breaks.
If inactive, ds-breaks repaired by more error-prone microhomology-mediated end joining (MMEJ).
Several syndromes associated with NHEJ defects (e.g. LIG4 syndrome / XLF-SCID), characterised by cellular radiosensitivity, microcephaly and severe combined immunodeficiency.
Provide some examples of cancer susceptibility syndromes caused by mutations in DNA damage response genes
Fanconi Anaemia (FANCA/C/G)
Ataxia Telangiectasia (ATM)
Xeroderma pigmentosum (XPC/ERCC2)
Nijmegen Breakage Syndrome (NBS)
Li-Fraumeni (TP53)
HNPCC (MLH1, MSH2/6)
HBOC (BRCA1/2)
Polyposis/FAP (MUTYH)
