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Abstract
Cells of organisms are constantly exposed to genomic insults such as oxidative stress from endogenous sources or environmental agents. Oxidative stress induces oxidative DNA damage such as apurinic/apyrimidinic (AP) sites, single-strand breaks (SSBs), and double-strand breaks (DSBs). Oxidative DNA damage is repaired primarily by base excision repair (BER) pathway as well as other DNA repair pathways. It is widely accepted that unrepaired oxidative DNA damage compromises DNA replication and transcription, leading to cancer and neurodegenerative disorders. As a cell-free biochemical model, Xenopus laevis egg extract has been utilized to investigate critical questions in the fields of DNA repair and DNA damage response pathways (Chapter 1). In my PhD thesis, I sought to elucidate how XRCC1 (Chapter 2) and Polymerase beta (Pol beta, Chapter 3), two protein crucial to the BER pathway, are involved in genomic integrity in Xenopus egg extracts. Here I report that when XRCC1 is depleted ATR-Chk1 signaling increases in Xenopus egg extracts following oxidative stress. When looking at the total amount of damage in the nucleus depletion of XRCC1 causes a statistically significant increase in the amount of DNA damage. XRCC1 is not, however, important for the repair of defined SSB plasmids in these extracts implying that oxidative DNA damage may be repaired in a BER independent manner. Despite this I show that XRCC1 can interact with APE2 in-vitro and plays a very minimal role for APE2’s exonuclease activity. In addition, ATR-Chk1 signaling following oxidative stress was decreased when Pol beta was removed. However, we can’t distinguish whether this Pol beta depletion phenotype is due to the absence of Pol beta, or co-depletion of integral proteins in the ATR-Chk1 pathway that interacts with Pol beta. When Pol beta is depleted, there is no significant increase in DNA damage following oxidative stress. Like XRCC1, Pol beta is dispensable for the repair of a defined SSB plasmid in Xenopus egg extracts. Instead, Pol alpha may be involved in the defined SSB plasmid repair. Taken together, these findings suggest that XRCC1 and Pol beta play distinct roles in the maintenance of genomic integrity in Xenopus egg extracts.