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Replicative and Transcriptional Bypass of Alkylated and Alpha-Anomeric Lesions

  • Author(s): Williams, Nicole Lynn
  • Advisor(s): Wang, Yinsheng
  • et al.
Abstract

Exposure to a variety of endogenous and exogenous sources of DNA damaging agents can lead to the formation of 104-106 DNA lesions per cell per day. These sources include reactive oxygen species (ROS), alkylating agents, ultraviolet (UV) light and various environmental chemicals that can interact with several positions of the DNA altering the structure, including the ring nitrogen and extracyclic oxygen atoms of the DNA bases. To counteract the deleterious effects of DNA damage, cells are equipped with machinery capable of quickly and efficiently repairing the damage, thereby maintaining genomic integrity. However, some DNA lesions are not efficiently repaired and have the potential to induce mutations and block replication and transcription machinery, ultimately disrupting the flow of genetic information. Understanding how DNA lesions perturb the flow of genetic information is important for determining how they contribute to various diseases, including cancer.

In Chapters 2, 3 and 4, we assessed the roles of translesion synthesis (TLS) DNA polymerase in the replicative bypass of various O2- and O4-alkylthymidine and -anomeric lesions in vitro by conducting primer extension and steady-state kinetic assays. These results provided important insights into which TLS polymerases are involved in the bypass of these lesions, and if the size and shape of the alkyl group adducted to the DNA differently affect the efficiency and fidelity of the polymerase-mediated bypass and what mutations are likely to arise during the replicative bypass.

In Chapter 5 we aimed to determine how the O2-alkylthymidine lesions affect the efficiency and fidelity of transcription in human cells. Additionally, we aspired to determine the pathways involved in the repair of these lesions through the use of isogenic cells where XPC or CSB proteins were depleted via the CRISPR/Cas9 genome editing method. These results offered new knowledge about the impacts of the various O2-alkylthymidine lesions have on transcriptional bypass efficiency, what mutations arise during transcriptional bypass and what repair pathways are involved in the repair of these lesions.

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