University of Sussex
Kakarougkas,_Andreas_David_William.pdf (32.58 MB)

The role of ATM signalling and its mediator proteins in DNA double strand break repair

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posted on 2023-06-08, 12:31 authored by Andreas David William Kakarougkas
Although most DNA double strand breaks (DSBs) are repaired by DNA nonhomologous end!joining (NHEJ), DSBs at heterochromatin (HC) regions undergo repair by homologous recombination (HR) in G2 phase. Repair of DSBs at HC regions requires ATM-dependent KAP1 phosphorylation and subsequent HC relaxation. The mediator proteins facilitate DSB repair at HC in G1 phase by retaining ATM and hence pKAP1 at DSBs until the completion of repair. In this thesis, I investigated the role of the mediator proteins in enabling DSB repair in G2 phase. I demonstrate that the mediator proteins are required for the slow component of DSB repair in G2, which represents HR. They also promote ATM-dependent pKAP1 formation in G2 as in G1. In addition, I have described a role for MDC1 in Rad51 loading and for RNF8 in DNA resection. Moreover, I demonstrate that BRCA1 overcomes an inhibitory barrier by 53BP1 to resection by promoting a G2!specific enlargement in 53BP1 foci during HR that involves 53BP1 repositioning to the foci periphery and vacation from the central core. RPA foci form in the core devoid of 53BP1. 53BP1 has opposing roles in HR; it creates a restrictive barrier to resection but promotes pKAP1 and HC relaxation. RAP80 also inhibits resection by binding to ubiquitylated histones at DSBs. I demonstrate that the DUB enzyme, POH1, is required to overcome the barrier posed to resection by RAP80 since its depletion leads to deficient 53BP1 vacation of the central core and deficient resection. BRCA1 and POH1 cooperate during G2 phase to promote resection and DSB repair by HR. Additionally; I investigated the role(s) of the chromatin remodelers BAF180 and CHD7 in transcriptional silencing following DSB induction, a process that requires ATM, RNF8 and RNF168. I demonstrate that deficient transcriptional silencing leads to a DSB repair defect at early times post IR.


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University of Sussex

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