Defense Date

2009

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Human Genetics

First Advisor

Kristoffer Valerie

Second Advisor

Lawrence Povirk

Abstract

This thesis investigates two separate features of nonhomologous end-joining (NHEJ) DNA repair: end processing, and DNA repair kinase signaling. DNA end processing was investigated in a mouse model of hereditary spinocerebellar ataxia with axonal neuropathy (SCAN1), a congenital neurodegenerative disease. SCAN1 is caused by a homozygous H493R mutation in the active site of tyrosyl-DNA phosphodiesterase (TDP1). To address how the H493R mutation elicits the specific pathologies of SCAN1 and to further elucidate the role of TDP1 in processing DNA end modifications, we generated a Tdp1 knockout mouse and characterized their behavior and specific repair deficiencies in extracts of embryonic fibroblasts from these animals. While Tdp1(-/-) mice appear phenotypically normal, extracts from Tdp1(-/-) fibroblasts exhibited deficiencies in processing 3'-phosphotyrosyl single-strand breaks and 3'-phosphoglycolate (PG) double-strand breaks (DSBs). Supplementing Tdp1(-/-) extracts with H493R TDP1 partially restored processing of 3'-phosphotyrosyl single-strand breaks, but with evidence of persistent covalent adducts between TDP1 and DNA, consistent with a proposed intermediate-stabilization effect of the SCAN1 mutation. However, H493R TDP1 supplementation had no effect on PG termini on 3' overhangs of DSBs; these remained completely unprocessed. Altogether, these results suggest that for 3'-PG overhang lesions, the SCAN1 mutation confers loss of function, while for 3'-phosphotyrosyl lesions, the mutation uniquely stabilizes a reaction intermediate. Furthermore, there is evidence that TDP1 also localizes to mitochondria, and mitochondrial DNA damage should not be excluded from significantly contributing to SCAN1 pathology. The effect of ATM signaling on NHEJ was investigated via a novel vector that allows for inducing I-SceI-mediated DNA DSBs that can then be analyzed for NHEJ repair events by fluorescence- and PCR-based methods. Using highly specific DNA kinase inhibitors and the repair cassette, we showed that inhibiting ATM reduced NHEJ by 80% in a U87 glioma model. Analysis of the PCR products from the NHEJ repair vector by PsiI restriction cleavage allowed for assessment of the fidelity of the NHEJ repair: inhibiting ATM reduced high-fidelity NHEJ by 40%. Together, these results suggest that ATM is critical for NHEJ of I-SceI DSBs and for high-fidelity repair, possibly due to ATM's effects on chromatin architecture surrounding the DSB.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

December 2009

Share

COinS