Investigation of DNA double-strand break-associated histone post-translational modifications using targeted mass spectrometry

Kozik, Zuzanna (2019) Investigation of DNA double-strand break-associated histone post-translational modifications using targeted mass spectrometry. Doctoral thesis (PhD), University of Sussex.

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DNA double strand breaks (DSBs) pose a major threat to the maintenance of genetic integrity. Cells have evolved response pathways to detect, signal and repair those lesions. Alterations in the factors involved in these pathways may lead to disease development, such as cancer. Several histone modifiers have previously been shown to be recruited to the sites of DSBs, but their role in the repair process still remains unclear. It has been proposed that the cellular response to DSBs leads to changes in phosphorylation, methylation, acetylation and biquitination and other post-translational modifications (PTMs) of histones at the site of damage. Some of these modifications are known epigenetic markers involved in maintaining cellular identity. It has been proposed that DSB-induced alterations to the epigenetic code introduce a potential window of opportunity for pathological changes to occur.
Here, I have developed a chromatin immunoprecipitation followed by mass spectrometry (ChIP-MS) method to enrich for mono-nucleosomes containing Ser139-phosphorylated H2AX (H2AX). I utilise targeted mass spectrometry to quantify histone PTMs associated with H2AX formed after ionising radiation (IR) damage of HEK293 cells, as well as wild type and ATM deficient 1BR fibroblasts. Surprisingly, few local changes in histone PTMs associated with H2AX containing mono-nucleosomes were found. A damage-dependent increase in H2A(X) lysine15 ubiquitination (H2A(X) K15Ub) was detected and I gained insight into the dynamics of this important PTM. We found that H2AX levels are maximal within 30 min of IR exposure after 3 Gy whilst H2A(X) K15Ub reaches maximal level at 4-8 h. A dose-response analysis revealed that whilst H2AX levels increase linearly with dose, the level of H2A(X) K15Ub peaks at ~3 Gy and is substantially diminished after 40 Gy, demonstrating that the response is not linear with dose and becomes saturated at higher doses. Furthermore, our preliminary data suggests that contrary to previous reports ATM-dependent late repairing DSBs are not enriched in constitutive heterochromatin marks. I discuss the clinical significance of these findings

Item Type: Thesis (Doctoral)
Schools and Departments: School of Life Sciences > Sussex Centre for Genome Damage and Stability
Subjects: Q Science > QH Natural history > QH0301 Biology > QH0426 Genetics
Depositing User: Library Cataloguing
Date Deposited: 14 Aug 2019 10:00
Last Modified: 14 Aug 2019 10:00

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