Defense Date

2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Pharmacology & Toxicology

First Advisor

Lawrence F Povirk

Abstract

DNA double-strand breaks (DSB) with chemically modified end-termini are the most significant lesions resulting from radio/chemotherapeutic intervention of cancer and non homologous end-joining (NHEJ) factor Artemis nuclease has been implicated in the repair of such breaks. To examine whether the resolution of terminally blocked DNA DSBs is the biologically relevant function of Artemis, Artemis deficient fibroblasts were stably complemented with wild type or an endonuclease deficient D165N mutant Artemis. Physiological levels of wild type (WT) Artemis completely restored DSB repair proficiency and resistance to γ-radiation, bleomycin, and neocarzinostatin. Cells expressing the D165N mutants remained as chemo/radiosensitive and as repair deficient as parental cells, with persistent γ H2AX and 53BP1 foci that increased in size 6-18 hour post irradiation. These persistent foci co-localized with DNA double strand break repair factor Mre11 and also with promyelocytic leukemia protein (PML). Further, in vitro studies have revealed that DNA-PK dependent Artemis endonucleolytic activity may play a role in the repair of commonly found oxidative base damage; 8-oxoguanine (8-oxoG), a hallmark of complex DSBs. However, majority of DNA DSBs are repaired in an Artemis independent manner, and recently discovered, DNA end-specific nuclease, Metnase is a candidate enzyme for repair of such breaks. To study the role of Metnase in resolution of 3ʹ-blocked termini, several substrates mimicking such breaks were constructed. A 3ʹ-phosphoglycolate moiety on longer overhangs (4 and 6 bases) altered specificity and stimulated Metnase-mediated cleavage of the terminal 3 nucleotides. However, an 8-oxoG residue at the single-strand/double-strand border did not affect specificity or extent of cleavage. Metnase preferentially cleaved ssDNA-overhang of a partially duplex substrate, and the cleavage increased with increase in length of 3ʹ-overhangs. A D483A mutation in Metnase completely abrogated Metnase cleavage activity towards DNA ends. These results suggest that Metnase may resolve oxidatively damaged DNA ends to facilitate repair while Artemis is required for the resolution of more complex DNA DSBs that persist for longer times and are not amenable to repair by other NHEJ factors.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

5-10-2012

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