Loss of DNA damage-induced G2 checkpoint control is associated with genome instability, tumour formation and the therapeutic response of tumours to genotoxic agents. The large 3418 residue protein encoded by BRCA2 – heterozygous germline mutations in which predispose to cancer - has recently been implicated in G2 checkpoint maintenance. However, the mechanistic basis of BRCA2’s role in the G2 checkpoint remains unknown. The overall aim of my research is to understand the mechanism by which BRCA2 regulates the G2 checkpoint. Domain mapping studies, using overlapping fragments encoding the full-length BRCA2 protein, carried out in our laboratory suggest that BRCA2’s function at the G2 checkpoint is mediated through regions that span BRCA2 amino acids (aa) 1-454 and aa 2438-2824. My research has focused on understanding how these two regions contribute to G2 checkpoint function through the interrogation of two novel interactions of BRCA2 mediated at these regions. My experiments have identified that BRCA2 interacts with the serine/threonine kinase ATR (aa 2438-2824) and the deSUMOylase SENP1 (aa 1-454). ATR is known to play a key role at the G2 checkpoint and my results identify that loss of BRCA2 leads to a reduction in ATR activity at sites of damage. This leads to a downstream attenuation in the phosphorylation of Chk1 – an important effector protein of the G2 checkpoint. BRCA2 is known to function in DNA repair and is recruited to sites of DNA damage, where it displaces RPA bound to exposed single-strand DNA. RPA is required to localise and activate ATR during the G2 checkpoint and therefore I hypothesise that BRCA2’s role at the checkpoint is to substitute for RPA and mediate ATR activation. I have also shown that SENP1 interacts with BRCA2 at a region within the N-terminus (aa 290-454). SUMOylation has been increasingly recognised as an important post-translational modification (PTM) in the context of the DNA damage response, and SENP1 is involved in reversing this PTM. I have shown that BRCA2 is SUMOylated. Moreover, I have shown that loss of SENP1 prevents BRCA2 from being recruited to chromatin in a timely manner after DNA damage and that SENP1 depletion exacerbates the loss of G2 checkpoint maintenance seen in BRCA2 deficiency. Taken together, this work reveals novel insights into the mechanism by which BRCA2 maintains genomic stability through enforcement of the G2 checkpoint. This new knowledge has the potential to translate into a better understanding of how mutations in BRCA2 may lead to cancer.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:753363 |
| Date | January 2018 |
| Creators | Ahmad, Syed Saif |
| Contributors | Venkitaraman, Ashok |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.repository.cam.ac.uk/handle/1810/278013 |
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