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Betts, Hannah (2024) Mechanisms of Replication Protein A-dependent R-loop resolution by the human DNA repair helicase, HelQ. PhD thesis, University of Nottingham.

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Abstract

To mitigate the devastating effects of unrepaired DNA damage, prokaryotes and eukaryotes alike have an abundance of mechanisms to correct potentially genome-destabilising insults as and when they arise. DNA repair helicases play an important role in coordinating this repair.

Of particular interest is the human DNA repair helicase HelQ – a lesser-characterized protein, whose deletion in mice manifests as an increased prevalence of ovarian tumours and infertility. HelQ is thought to stabilize stalled replication forks in a recombination-independent manner. This includes stalled forks caused both by interstrand crosslinks and R-loops – RNA:DNA hybrids which in excess contribute to genome instability. In this respect, HelQ plays an anti-cancer role for the human genome. HelQ is known to interact with Replication Protein A (RPA), a ssDNA binding protein involved in homologous recombination and other repair mechanisms, known to stimulate the helicase activity of HelQ.

To investigate the function of HelQ in the context of R- loops resolution, we present immunocytochemical data indicating a possible but unverified link between HelQ expression and nuclear R-loop levels. Therefore, we further employ dot-blot based immunoassays to examine the factors contributing to R-loop removal, and report that HelQ is strongly associated with R-loop homeostasis through an ATP dependent and RPA-stimulated R-loop clearing role.

We further present biochemical data examining the structure of the N-terminal region of HelQ (N-HelQ). We provide supporting evidence that this protein fragment exhibits considerable intrinsic disorder. Experimentation with nuclear magnetic resonance (NMR) with N-HelQ and RPA suggest that N-HelQ is responsible for the HelQ:RPA interaction.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Soultanas, Panos Williams, Huw
Keywords:	DNA, helicases, HelQ
Subjects:	Q Science > QP Physiology > QP501 Animal biochemistry
Faculties/Schools:	UK Campuses > Faculty of Science > School of Chemistry
Item ID:	77398
Depositing User:	Betts, Hannah
Date Deposited:	24 Jul 2024 04:40
Last Modified:	24 Jul 2024 04:41
URI:	https://eprints.nottingham.ac.uk/id/eprint/77398

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