Abstract
Homologous recombination (HR) is one the main mechanisms for repairing DNA double strand breaks (DSBs), as well as being a key downstream step in the repair of other DNA lesions, such as interstrand crosslinks (ICLs). The importance of HR for maintaining genome stability is underscored by the observation that loss-of-function mutations in HR factors are commonly seen in human cancers. In this thesis, I report the use of a key hallmark of HR-deficient cells, namely synthetic lethality with the poly-ADP ribose polymerase (PARP) inhibitor olaparib, as a means of screening for new HR factors. In this manner, I identify the E2 ubiquitin conjugating enzyme UBE2S as a novel factor required for olaparib resistance, and confirm its direct involvement in DNA repair by showing that it rapidly accumulates at sites of DNA damage. This work highlights UBE2S as a potentially useful new biomarker for treatment of cancers with olaparib.In the second half of this thesis, I identify the E3 ligase RFWD3 as a protein required both for efficient HR and ICL repair. Defects in the repair of ICLs are associated with the genome instability syndrome Fanconi anemia (FA) and, consistent with this, RFWD3 mutations were recently identified in a new FA subtype. I demonstrate that the FA-associated I639K mutation, located in the WD40 repeats of RFWD3, abolishes interaction with its substrate RPA and consequently prevents recruitment of RFWD3 to sites of ICLs. Moreover, point mutations in RPA32 that prevent RFWD3 binding also disrupt ICL repair, and unloading of RPA from ssDNA at sites of ICLs is perturbed in RFWD3-deficient cells. Furthermore, loss of RFWD3 leads to defective HR, likely due to the persistence of RPA on ssDNA. Together these data reveal critical roles for RFWD3 in the repair of damaged DNA and the maintenance of human health.
| Date of Award | 2017 |
|---|---|
| Original language | English |
| Sponsors | Medical Research Council |
| Supervisor | John Rouse (Supervisor) |