PARP/XRCC1 surveillance of the human genome

Gittens, William (2018) PARP/XRCC1 surveillance of the human genome. Doctoral thesis (PhD), University of Sussex.

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Abstract

DNA single strand breaks (SSBs) are one of the most common lesions to genomic DNA, arising from various endogenous and exogenous sources. Single strand break repair (SSBR) constitutes a biochemical pathway whereby SSBs are detected, enzymatically processed and ligated. Whilst the general mechanisms of SSBR are relatively well described in vitro, there are remaining questions concerning how the pathway operates in vivo. For example, an early step in SSBR is thought to be the poly(ADP-ribose) polymerase (PARP)-dependent modification of SSB-proximal proteins with ADP-ribose, which is a signal for the recruitment of downstream repair factors, including the central scaffold XRCC1. Yet, the presence of multiple DNA-dependent PARP genes (PARP1, PARP2 and PARP3) has caused confusion regarding their specific roles in SSBR. This thesis potentially clarifies some contentious aspects of PARP function in the repair of SSBs induced by reactive oxygen species (ROS) and by Topoisomerase 1 (Top1). By employing PARP1-/-/PARP2-/- cells generated herein using CRISPR-Cas9 technology, in combination with preextraction immunofluorescence imaging and high-content analysis, I demonstrate that both PARP1 and PARP2 contribute towards ROS-induced ADP-ribosylation and XRCC1 chromatin-localization, but that in response to Top1-SSBs, these functions are specifically supported by PARP1 alone. Furthermore, using TDP1-/- and XRCC1-/-/TDP1-/- cells also generated herein, I characterize a striking hyper-ADP-ribosylation phenotype in response to Top1-SSBs. The clinical significance of this was confirmed by co-workers, who observed a similar phenotype in an XRCC1-deficient patient, where mutations in XRCC1 underlie a novel cerebellar neurodegenerative disease. This phenotype could be utilized in future to screen for genes with novel functions in SSBR. Finally, I investigate the functional implications of disrupted SSBR genes for rates of repair and cellular viability using alkaline single-cell electrophoresis and clonogenic survival assay. In doing so, I unexpectedly discovered that deletion of PARP1 suppresses CPT-induced comet tail moments of WT and XRCC1-/- cells.

Item Type: Thesis (Doctoral)
Schools and Departments: School of Life Sciences > Sussex Centre for Genome Damage and Stability
Subjects: Q Science > QP Physiology > QP0501 Animal biochemistry > QP0620 Nucleic acids > QP0624 Deoxyribonucleic acids > QP0624.5.A-Z Special topics, A-Z > QP0624.5.S73 Stability
Depositing User: Library Cataloguing
Date Deposited: 30 May 2018 11:55
Last Modified: 30 May 2018 11:55
URI: http://sro.sussex.ac.uk/id/eprint/76255

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