DOI
https://doi.org/10.25772/KQ3H-CN65
Author ORCID Identifier
https://orcid.org/0000-0002-3787-5930
Defense Date
2018
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Pharmacology & Toxicology
First Advisor
Lawrence F Povirk
Abstract
Clustered damage to DNA is a signature mark of radiation-induced damage, which involves damage to the nucleobases and/or DNA backbone. Double-strand breaks created by damaging agents are detrimental to cell survival leading to chromosomal translocations. Normal cells employ Non-homologous end-joining because of its faster kinetics, to suppress chromosomal translocations. However, the presence of complex DNA ends constitutes a significant challenge to NHEJ. Location of Thymine glycol (Tg) at DSB ends was a potential hindrance to end joining. The substrate with Tg at the third position (Tg3) from the DSB joined better than when present at the fifth position (Tg5). However, hNTH1 assay showed Tg5 to be a better substrate than Tg3 for BER, potentially explaining the increased Tg removal and decreased end joining of Tg5 in extracts. Nonetheless, there appeared to be no preference in the susceptibility of 5’-Tg substrates with Tg at the second and third positions from DSB ends.
Polynucleotide kinase phosphatase is crucial in restoring the 3′ hydroxyl, and 5′ phosphate ends at strand breaks. No other enzyme is known to possess PNKP’s activity in mammalian cells at DSBs. Experiments done with PNKP knockout cells have shown some activity similar to PNKP, which appeared to be a part of NHEJ and was not pharmacologically inhibited by PNKP inhibitor. Additionally, core NHEJ factors XRCC4 and XLF influenced the activities of PNKP.
Overall, these experiments suggest that Tg repair is dependent on the position from DSB and an alternative enzyme processes 3′- PO, and 5′-OH ends.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
2-13-2018