Defense Date

2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Pharmacology & Toxicology

First Advisor

Lawrence F. Povirk

Abstract

DNA double-strand breaks induced by ionizing radiation are often accompanied by ancillary oxidative base damage that may prevent or delay their repair. In order to better define the features that make some DSBs repair-resistant, XLF-dependent nonhomologous end joining of blunt-ended DSB substrates having the oxidatively modified nonplanar base thymine glycol (Tg) at the first (Tg1) , second (Tg2), third (Tg3) or fifth (Tg5) positions from one 3’ terminus was examined in human whole-cell extracts. Tg at the third position had little effect on end-joining even when present on both ends of the break. However, Tg as the terminal or penultimate base was a major barrier to end joining (>10-fold reduction in ligated products) and an absolute barrier when present at both ends. Dideoxy trapping of base excision repair intermediates indicated that Tg was excised from Tg1, Tg2 and Tg3 largely if not exclusively after DSB ligation. However, Tg was rapidly excised from the Tg5 substrate, resulting in a reduced level of DSB ligation, as well as slow concomitant resection of the opposite strand. XLFL115D mutant completely eliminates ligation of all five substrates and previous X‑ray crystallography shows that XLF binds to XRCC4 via a “leucine lock” motif wherein L115 of XLF slips into a hydrophobic pocket in XRCC4. This makes the XRCC4-XLF interaction a good target to develop peptide inhibitors in order to radiosensitize breast tumor cells that are dependent on NHEJ to repair their DSBs after ionizing radiation exposure. Using mRNA display, we created a diverse library of 870 billion unique peptide sequences. After seven rounds of in vitro selection, the eluted fusions were cloned and sequenced. The results showed homology of sequences of five main families. We have selected representative peptides from those families (Pep 7.1-7.5), and several were chemically synthesized. However, none of these significantly inhibited XLF-dependent end joining in whole-cell extracts. Overall, the results suggest that promoting ligation of DSBs with proximal base damage may be an important function of XLF, but that Tg can still be a major impediment to repair, being relatively resistant to both trimming and ligation. The effectiveness of XLF-XLRCC4 inhibitors in blocking nonhomologous end joining remains to be determined.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

7-20-2015

Available for download on Saturday, July 18, 2020

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