20.06.10 DNA damage response and cancer Flashcards
1
Q
Examples of DNA damage
A
- Environment
- Genotoxic agents (endogenous, exogenous)
- Intrinsic instability of DNA
- Damage during DNA replication (misincorporation of nucleotides)
2
Q
What happens if DNA damage is not repaired
A
- Causes blockages in transcription and replication
- Mutagenesis
- Cellular toxicity
3
Q
What are the 4 DNA repair mechanisms
A
- Mismatch repair (MMR)
- Base excision repair (BER)
- Nucleotide excision repair (NER)
- Direct repair: Repair of double strand breaks using homologous recombination and non homologous end joining
4
Q
What is MMR
A
- Mismatch repair pathway
- Recognises erroneous insertions/deletions and misincorporations of bases that arise during DNA replication and recombination.
- Uses 3 essential proteins MutS, MutH (in E.coli) and MutL
- Defects in the MMR pathway result in damaged DNA proceeding through the cell cycle, increasing the mutation rate and the risk of oncogenesis.
5
Q
What genes encode MutS and MutL
A
- MutS: MSH2, MSH6
- MutL: MLH1, MLH2, MLH3, PMS1, and PMS2
- Commonly mutated in Lynch syndrome, leading to MSI (microsatellite instability)
6
Q
What is BER
A
- Base excision repair pathway
- Removes small non-helix-distorting base lesions from the genome and repairs single-strand breaks
- Base damage can be caused by ROS, ionising radiation, X-rays and certain chemicals
- e.g. MUTYH mutations cause MAP (MUTYH associated polyposis)
7
Q
Steps of MMR pathway
A
- MSH2 and MSH6, recognise and bind to the mismatched base(s).
- MLH1 and PMS2 are then recruited and the DNA strands are cleaved
- The mismatched base is removed by an exonuclease (EXO1) and the gap is filled in by DNA polymerase.
8
Q
Steps of BER pathway
A
- Damaged base is removed by different DNA glycosylases (e.g. MUTYH) resulting in an abasic site.
- abasic site is a substrate for AP (apurinic/apyrimidinic) endonuclease (APE1), which converts it into a SSB
- The break is then repaired by short (major)- or long (minor)-patch BER.
- The short-patch pathway involves polymerase β, which removes a 5′-deoxyribose moiety and inserts a single base patch that is sealed by DNA ligase III.
- The scaffold protein, XRCC1, interacts with many of the pathways components and functions to anchor them to the substrate through successive stages of BER.
9
Q
What is NER (nucleotide excision repair)
A
- Mechanism for repair of bulky DNA lesion (e.g. thymine dimers) caused by UV radiation, ROS, genotoxic chemicals
- 2 classes:
1. Global excision repair (GER) which repairs all DNA and
2. Transcription-coupled repair (TCR) where DNA undergoing transcription is repaired.
10
Q
Steps of NER pathway
A
- Global excision repair= where the XPC protein scans the genome constantly and recognises damage.
Or
Transcription-coupled repair= recognises the stalling of RNA polymerase during transcription due to the presence of DNA damage. - DNA duplex unwinds by transcription factor IIH (TFIIH). Another protein, XPA, confirms the presence of DNA damage and if it is not detected NER is aborted.
- Lesion removed and gap filled using low fidelity polymerases
11
Q
Review of the repair of double strand DNA breaks
A
- dsDNA breaks due to ROS, ionising radiation, genotoxic chemicals
- dsDNA breaks could lead to chromosomal fragmentation, translocations and deletions.
- 2 mechanisms
1. Homologous recombination (HR)
2. Non-homologous end joining (NHEJ) - Disorders due to defects in this pathway include Bloom syndrome, BRCA1/2-associated breast/ovarian cancers, Nijmegen breakage syndrome, LIG4 syndrome.
12
Q
Review of homologous recombination
A
- Only occurs where there is a homologous sister chromatid (i.e. in G2 phase in dividing cells)
1. Uses a homologous stretch on sister chromatid (>300bp)
2. single strand overhangs are created in a process that is likely mediated by the MRN (Mre11/Rad50/Nbs1) complex
3. Rad51, Rad52, and RPA genes recognise overhangs
4. DNA synthesis
13
Q
Review of Non-homologous end joining (NHEJ)
A
- Uses short homologous DNA sequences (microhomologies) present on single-stranded overhangs on the ends of double strand breaks to guide repair
- two ends of the broken helix are brought together to form a synaptic complex (2 DNA ends, 2 Ku70/80 and 2 DNA PKCS molecules)
- Repair of break by DNA ligase IV/ Xrcc4 complex
- Less accurate and can give rise to deletions.
14
Q
What is translesion DNA synthesis
A
- DNA damage tolerance process that allows the DNA replication machinery to replicate past DNA lesions, bypassing stalled replication forks. At the cost of a high error rate
- Uses low fidelity polymerases e.g. zeta and iota polymerases.
- Translesion synthesis is a major source of DNA damage induced mutagenesis.
15
Q
What advantages are there for the high error rates in translesion synthesis
A
-Contributes to diversity, e.g of immunoglobulins, to assist in the recognition of numerous foreign antigens.
