Defense Date

2015

Document Type

Thesis

Degree Name

Master of Science

Department

Pharmacology & Toxicology

First Advisor

Dr. Lawrence F. Povirk

Abstract

DNA double-strand breaks containing unligatable termini are potent cytotoxic lesions leading to cell death or growth arrest. Artemis, which is associated with the Non-Homologous End Joining (NHEJ) pathway, is the major end processing nuclease that resolves unligatable termini, especially the 3′ blocks, by nucleolytic trimming. Tyrosyl-DNA Phosphodiesterase 1 (TDP1) is an enzyme which is biochemically competent in 3′-phosphoglycolate processing. The purpose of this study is to investigate if TDP1 is an end-processing enzyme involved in the NHEJ pathway. Clonogenic Survival assays using shRNA-mediated TDP1 knockdown and Artemis knockout (Artemis-/-) in HCT116 cells showed increased sensitivity to Neocarzinostatin (NCS) and Calicheamicin, radiomimetic drugs that produce 3′-phosphoglycolate-terminated double-strand breaks. Thus, a cell line with combined deficiency in Artemis and TDP1 was generated by infecting Artemis-/- single mutants with a lentivirus expressing a TDP1 shRNA. Positive clones were screened for maximum TDP1 knockdown which was found to be 10X. Clonogenic survival assays carried out on shTDP1 & Artemis-/- single mutants and the Artemis-/-.shTDP1 double mutants showed similar sensitivity to Calicheamicin and NCS. Immunofluorescence studies on Art-/- and Art-/-.shTDP1 mutants also showed a similar increase in persistent 53BP1 foci, a measure of DNA damage, after treatment with NCS. Cell cycle analysis studies showed all these mutants arrest in G1 phase of the cell cycle after treatment with NCS. Thus, taken all together, surprisingly, these experiments suggest that TDP1 functions are epistatic with Artemis in the NHEJ pathway for repair of Calicheamicin- and NCS-mediated DNA damage.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

5-4-2015

Available for download on Saturday, May 02, 2020

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