Homologous recombination: strand invasion Flashcards
(24 cards)
What is strand invasion?
Where the 3’ ssDNA produced by resection invades a homologous sequence in the repair template forming a D loop structure through a reaction called strand exchange
What are the main proteins involved in strand invasion? (2)
- Rad51 (and Rad52) for initial invasion
- BRCA2 (and BRCA1 and Rad51) for strand exchange
What does live cell imaging show about RPA and Rad51 binding to DNA?
RPA binds to DNA damage first, followed by Rad51 (sequential recruitment)
What is the model of RAD51 mediated strand invasion? (4)
- RPA binds the 3’ overhangs following resection to prevent self-folding of ssDNA which would block association of repair proteins e.g. Rad51 and Rad52
- Rad52 can bind RPA/ssDNA and displace RPA which is important for Rad51 to bind ssDNA
- Rad52 recruits Rad51 to 3’ ssDNA
- Rad51 triggers strand invasion
What are the risks associated with BRCA1 and BRCA2? (5)
- Tumour suppressors which account for 5% of breast cancers in the USA
- BRCA2 families also have increased risk of male breast, pancreas and prostate cancers
- One mutated copy is inherited and loss/mutation in the second allele leads to LOH in two hit hypothesis
- Mutation of a single allele may be sufficient for increased tumour risk
- LOH is seen in some but not all cancer cells so may be a later event rather than initiating
How do BRCA2 and Rad51 differ in DNA binding preference? (2)
- BRCA2 specifically binds to ssDNA
- Rad51 has no selective binding of ssDNA and dsDNA
How does BRCA2 influence Rad51 DNA binding activity?
BRCA2 greatly increases Rad51 preference for ssDNA binding
How does BRCA2 influence strand exchange? (3)
- Incubate 3’ ssDNA with BRCA2 and Rad51, then add dsDNA
- If strand exchange occurs, a larger product should be formed where one of the dsDNA strand joins with the 3’ ssDNA tail, leaving one of the dsDNA strands free
- BRCA2 acts by targeting Rad51 to ssDNA over dsDNA, enabling Rad51 to trigger strand invasion
What is the model so far of HR from resection to invasion? (6)
- Mre11 complex/CtIP bind DSB ends and recruit BRCA1 to start resection
- Exo1 or DNA2 carries on further resection
- RPA binds the 3’ overhangs to prevent self-folding of ssDNA which would block repair protein recruitment
- Rad52 binds RPA/ssDNA and displaces RPA
- BRCA2 binds and recruits Rad51 to 3’ ssDNA
- Rad51 triggers strand invasion
What is the DNA damage response (DDR)? (3)
- DNA damage and activates kinases ATM (DSBs) and ATR (SSBs and replication stress)
- ATM and ATR activate CHK2 and CHK1 respectively and p53
- Cell cycle is halted to allow the cell to decide between repair or apoptosis
Why is the DDR important to cancer? (2)
- DNA repair is a barrier to maintain genomic stability and suppress tumorigenesis
- Defects in HR lead to use of error prone pathways causing accumulation of mutations and chromosomal translocations
What is the oncogene induced DNA replication stress model for sporadic cancer? (4)
- DNA repair genes are found to be mutated at a later stage of tumorigenesis in sporadic cancers vs inherited, after oncogene activation
- Replication stress triggers the DDR which works to maintain genomic stability
- Activated growth signalling in cancer increases replication stress which increases the DDR in pre-cancerous lesions
- This creates a selective pressure which pushes the cancer cell to try and inactivate DDR, resulting in a mature tumour
How can DNA damage be combined with existing therapeutics? (3)
- Classical cancer therapies work by inducing high levels of DNA damage and thus cell death
- Inhibitors of the DDR can sensitise to chemo and radiotherapy
- Issue is targeting this to cancer cells e.g. ATM has various normal functions so ATM inhibitors would be very damaging to healthy cells
What HR defects are commonly seen in tumours? (3)
- HR defects are most frequent in ovarian and breast cancer, followed by pancreatic and prostate
- Biallelic inactivation of BRCA1, BRCA2, RAD51C or PALB2 are the most common genetic cause of HR defects (each causes what is classed as a BRCA-like phenotype)
- Inactivation is predominantly associated with LOH
How is Rad51 impacted in cancer? (3)
- Rad51 protein is elevated in many cancer cell lines and primary tumours
- Overexpression can result in improper and hyper-recombination, contributing to genomic instability
- May drive regular cells towards neoplastic transformation or contribute to cancer progression, metastasis and drug resistance
Why is Rad51 hard to study in the lab?
Double knockouts are lethal
How could Rad51 be targeted by cancer therapies? (3)
- Rad51 depletion is synthetic lethal with PARP1/2, DNA-PK, p38 MAPK pathway (shows central role in DNA repair)
- Rad51 depletion sensitises to chemo and radiotherapy
- A clinical trial targeting Rad51 is currently active
How could Mre11 be targeted by cancer therapies? (3)
- Alterations of the MRN complex are linked to treatment resistance
- Could be inhibited in the lab with siRNA, CRISPR, small molecule inhibitors, Mirin (inhibits MRE11 exonuclease activity)
- MRE11 defects are synthetic lethal with PARP in model systems
What is neuroblastoma? (2)
- Childhood cancer of the nervous system commonly starting from one of the adrenal glands
- Highest cause of cancer deaths in children and infants
What categories can neuroblastoma be split into? (2)
- MYCN amplification and no amplification
- MYCN amplification associated which much poorer survival
What is MYCN? (6)
- Transcription factor involved in various processes such as p53 activation, angiogenesis etc
- Amplification in neuroblastoma correlates with rapid progression, early chemo resistance and poor prognosis
- MYC family includes c-MYC, MYCN, MYCL
- MYCN binds MYC E-boxes in promoters and enhancers to regulate transcription
- MYCN has homology to c-MYC but have differential expression during development (MYCN more kidney and nervous system, c-MYC more spleen and liver)
- c-MYC is shown to induce replication stress so may also be true for MYCN
How is MYCN linked to MRE11 in neuroblastoma? (2)
- Mirin decreases survival in MYCN amplified cell lines by inducing increased DNA damage and cell death
- Suggests that MYCN interacts with MRE11 which is important for dealing with DNA damage and cell death when MYCN is amplified
What is the model for the effects of MRE11 inhibition on MYCN overexpression tumours? (4)
- MYCN amplification causes increased DNA replication and proliferation which causes increased replication stress
- This triggers the DDR which activates p53 and cancer cell death
- To overcome this, MYCN interacts with MRE11 which regulates replication stress and DNA damage so it is at a level which evades p53 activation and allows the cancer cell to survive
- Therefore if you inhibit MRE11 with Mirin, MYCN is no longer regulated and the resultant DNA damage is enough to activate p53 and cause cancer cell death
Is MRE11 synthetic lethal with PARP? (2)
- Any defect in HR will be synthetic lethal with PARP
- Can use this to generate a HR deficient phenotype for treatments
