Publication Date

April 2018

Advisor(s)

Donald Oliver

Major

Molecular Biology&Biochemistry

Language

English (United States)

Abstract

Every day, our DNA is damaged by various phenomena, including environmental factors and chemical agents. One of the most cytotoxic forms of damage are DNA double-strand breaks (DSBs). DSBs are termed cytotoxic because normal cells are unable to tolerate unrepaired DSBs, and therefore undergo apoptosis. Because DSBs sever both strands of duplex DNA, no complementary strand is available to serve as a template for repair. Thus, in order for cells to repair DSBs and avoid apoptosis, they are likely to rely on either homologous DNA from the sister chromatid, which serves as repair template DNA, or on cell cycle checkpoint mechanisms that arrest cell-cycle progression and activate other DNA repair pathway mechanisms. If a cell suffers a DSB, it is likely to restore its genomic integrity by employing one of two major pathways: Classical Non-Homologous End Joining (C-NHEJ) and Homologous Recombination (HR). HR, a recombination-dependent pathway, is active in the S and G2 phases of the cell cycle and utilizes homologous DNA from the sister chromatid for repair. However, in C-NHEJ, blunt-DSB ends are ligated together (independently of end-sequence homology) during any phase of the cell cycle. Contrastingly, if lesions are encountered during the replication of DNA, then cells exploit a different pathway termed Translesion Synthesis (TLS). In this pathway, the lesion is not repaired, but tolerated by specialized TLS polymerases that assist in DNA damage bypass. More specifically, a polymerase switch occurs where the normal replicative polymerase is switched off, and a specialized, TLS polymerase is switched on. It is this specialized polymerase that promotes the bypass of lesions. One of the specialized TLS polymerases, DNA Pol ?, is unique in that it exists as a heterodimer of two different proteins, Rev3 and Rev7. These proteins create a structural interface for the binding of a different protein, Rev1 (Hara et al. 2010, 12299); moreover, recent studies suggest that additional Rev7-mediated interactions with Rev1 and Rev3 are responsible for the tolerance of DNA damage, where Rev7 functions as an adapter protein, recruiting Pol ? to lesion sites. This one subunit of Pol ?, Rev7, is not only an important protein in TLS, but also functions in targeted, DSB repair pathway choice, where it plays a role in the suppression of DSB end resection and therefore the suppression of HR (Boersma et al. 2015, 537). Studies make clear that an absence of Rev7 results in elongated 3? telomere overhangs, suggesting that Rev7 controls DNA repair at telomere ends and DNA breaks by inhibiting 5? end resection (Boersma et al. 2015, 537). Additionally, studies show that Rev7 accumulates at uncapped telomere ends, and acts downstream of early signalers 53BP1 and RIF1. Thus, Rev7 is involved in promoting C-NHEJ by suppressing HR, and DSB-end resection downstream of RIF1. The data presented in this thesis suggest that while Rev7 is involved in DNA repair and tolerance mechanisms, it is regulated by a different protein, thyroid hormone receptor interacting protein 13 (TRIP13). The overexpression of TRIP13 in several cancer types induces chemoresistance in cancer cells (Banerjee et al. 2016, 10726). Our central hypothesis is that TRIP13 performs various roles in DNA repair through its interactions with its likely substrate, Rev7, whereby TRIP13 negatively modulates Rev7 as a method to upregulate HR, and downregulate either TLS and/ or C-NHEJ.

Available for download on Monday, April 15, 2019

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