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Investigating the role of H2A.X di-phosphorylation in the DNA Damage Response

Smith, Nicola (2017) Investigating the role of H2A.X di-phosphorylation in the DNA Damage Response. Master of Science by Research (MScRes) thesis, University of Kent,. (KAR id:63932)

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Abstract

One of the earliest responses to DNA double strand breaks is phosphorylation of the C-terminal tail of histone variant H2A.X at position S139. The phosphorylated form (?H2A.X) is a marker of DNA damage and promotes DNA damage repair (DDR). Recent work showed that H2A.X can also be phosphorylated at position Y142, and that increasing Y142 phosphorylation by siRNA knockdown of the Eyes Absent (Eya) phosphotyrosine phosphatases (which remove Y142 phosphates) leads to increased apoptosis in response to DNA damage. This led to the proposal that an epigenetic "switch" between mono-?H2A.X (S139 only phosphorylated) and di-?H2A.X (S139 and Y142 both phosphorylated) directs the choice between DNA repair vs apoptosis in response to DNA damage. In this study, we sought to test this "switch" hypothesis by testing for cooperativity between the DNA damaging drug, Bleomycin, and Benzbromarone - a pharmacological inhibitor of Eya phosphatases. Although little cooperativity between Bleomycin and Benzbromarone was found in cell proliferation assays, live-cell imaging with fluorescent markers of apoptosis showed an increase in cell death. Moreover, by using novel antibodies that specifically recognise the various phosphorylated forms of H2A.X, immunochemical staining revealed that di-?H2A.X is located adjacent to and "flanks" ?H2A.X in DDR foci, and is interspersed with ?H2A.X in apoptotic cells. Therefore, contrary to the "switch" hypothesis being the singular function of di-?H2A.X, these results suggest an additional model for the role of di-?H2A.X in DDR, in that it may also act as a limiting factor in the rate of expansion of ?H2A.X repair foci.

Item Type: Thesis (Master of Science by Research (MScRes))
Thesis advisor: Ellis, Peter J.I.
Thesis advisor: Michaelis, Martin
Uncontrolled keywords: DNA Damage Response; (DDR) Histone; H2A.X; ?H2A.X; Phosphorylation Di-phosphorylation; Serine 139 (Ser139) Tyrosine 142 (Tyr 142) Apoptosis
Divisions: Faculties > Sciences > School of Biosciences
SWORD Depositor: System Moodle
Depositing User: System Moodle
Date Deposited: 10 Oct 2017 14:10 UTC
Last Modified: 22 Jan 2020 04:10 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/63932 (The current URI for this page, for reference purposes)
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