Defense Date

2014

Document Type

Thesis

Degree Name

Master of Science

Department

Pharmacology & Toxicology

First Advisor

Lawrence F. Povirk

Abstract

DNA double strand breaks (DSBs ) are extremely toxic to cells because they can lead to genomic rearrangements and even cell death. Two main pathways can repair DSBs: the homologous recombination repair (HRR) pathway and the non-homologous end-joining (NHEJ) pathway. NHEJ is the primary repair pathway in mammalian cells. HRR repairs single strand breaks (SSBs) or DSBs, mostly during late S phase and G2 phase of the cell cycle, by using an undamaged copy of the DNA sequence, and is therefore largely error-free, while the NHEJ pathway repairs DSBs without the requirement for sequence homology, may be error-free or error-prone, and is most active during G1 phase. Thymine glycol (Tg), the most common oxidation product of thymine. It produced endogenously as a consequence of aerobic metabolism or via exogenous factors such as ionizing radiation (IR), it is one of the predominant types of base modifications produced by ionizing radiation. Due to clustering of radiation – induced ionizations, Many DSBs induced by ionizing radiation bear damaged bases, including (Tg) moieties at or near the DSB ends that may interfere with subsequent gap filling and ligation. Artemis is a nuclease that is involved in the processing of termini during repair of DSBs and in modifying termini of complex DSBs. It has 5′–3′ exonuclease activity specific for single- stranded DNA, but, in the presence of DNA-PK, Artemis demonstrates endonuclease activity that is utilized in the removal of 3′ phosphoglycolate termini and 5′ overhangs, in the shortening of 3′ overhangs at DSBs, and in the opening of hairpin ends. To assess the ability of NHEJ to rejoin DSBs accompanied by Tg lesions and to elucidate the aspects of the possible role of Artemis in DSB repair, linearized plasmids with Tg either at the 3’ terminus of a blunt end (designated Tg1) or three or two bases from the end (Tg3 and Tg2, respectively), were subjected to a repair assay using XRCC4-like factor (XLF) deficient cell extracts, with or without the addition of XLF and/or Artemis, EndoIII and ddTTP. The data indicated that, the cell extract could ligate the plasmids with Tg1 and Tg2 with extremely low efficiency but could repair plasmid with Tg3 as efficiently as unmodified plasmid. In addition, Plasmids with Tg1and Tg2 were treated with Endonuclease III and ddTTP to test whether the end joining occurred before or after Tg removing, neither one had any effects on plasmids with Tg1. However, plasmids with Tg2 showed reduced in the intensity upon treatment with Endonucleases III and ddTTP, which suggested some ligation occur while Tg still present. In Artemis reaction, substrate with Tg2 and Tg3 could stimulate Artemis mediated trimming but not Tg1. Addition of EndoIII or ddTTP to plasmid with Tg3 resulted in a significant decrease in the intensities of the bandsrepresenting ligated products compared to XLF alone, suggesting that in some of the ligated products Tg are still present, while in others Tg had been removed and replaced by polymerization with normal nucleotides. Taken together, our results indicated that cell extract could ligate the plasmid with Tg located at the third base to DSB with high efficiency compared to plasmids with Tg1 and Tg2 which apparently this ability was severely inhibited when it located at or in the second position to DSB ends. Moreover, Artemis is also capable of trimming of thymine glycol at the second or third position from DSB ends with limited capability but inhibited by the presence of thymine glycol at the break site.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

8-18-2014

Available for download on Saturday, August 17, 2019

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