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Degree

Doctor of Philosophy, Ohio State University, Pharmacy, 2005.

Abstract

The primary focus of my thesis is to understand the effects of integrin cell adhesion receptor engagement on mouse lung endothelial cell (MLEC) DNA repair pathways and chromatin structural proteins. Engagement of Beta1 integrin receptors protects MLEC DNA from the genotoxic effects of bleomycin (BLM). Here we explored the impact of integrin engagement on chromatin structure. Integrin-engagement increased histone H3 acetylation and association of acetyl-histone H3 and histone H1 with DNA. Integrin protection from BLM-induced DNA damage requires poly(ADP-ribose) polymerase-1 (PARP-1). We investigated key components of single-strand break repair and base excision repair (BER). Integrin engagement reduced the ability to immunoprecipitate Lig3alpha and XRCC1, and decreased the KM value for DNA ligase activity. We have shown BLM dose-dependently increased double-strand DNA break marker gamma-H2A.X in fixed MLEC. Integrin-engagement reduced BLM-dependent gamma-H2A.X staining. Using small inhibitory RNA, integrin engagement-dependent decreases in BLM-induced gamma-H2A.X and single-strand breaks required BER enzyme Lig3alpha but not DNA ligase I (Lig1). Overall we show that integrin engagement may be operating through DNA repair systems to reduce the overall DNA damage in MLEC. Alterations in chromatin structure through “channeling” of DNA damage to Lig3alpha- and PARP-1-dependent repair may be the primary function of integrin engagement in the suppression of DNA breakage. Acetylation of core histones relaxes chromatin structure, decreasing histone tail interactions with DNA and regulating gene transcription and possibly DNA damage. Here we studied the effects of trichostatin A (TSA), a histone deacetylase inhibitor, on BLM-induced DNA damage in MLEC nuclei. In the presence of TSA, 15 minutes BLM treatment enhanced DNA breaks while 45 minutes BLM treatment decreased DNA breaks. The sequential recruitment of DNA repair enzymes to chromatin-bound DNA damage sites is essential for efficient repair. We hypothesized that BER proteins, PARP-1 and Lig3alpha preferentially interact with histones following DNA damage and enhancement or decrement of post-translational modifications on histones affects these interactions. BLM increased Lig3alpha and auto-modified PARP-1 association with histone H3. The results suggest DNA damage increases association of repair proteins with histones H1 and H3. Therefore, chromatin alterations may facilitate DNA repair through discrete, localized changes in histone-DNA interactions.

Subject Headings

Health Sciences, Pharmacy

Keywords

µg; DNA; BLM; MLEC; histone; PARP-1; integrin

Advisor

Dale G Hoyt

Pages

315p.

Document number: osu1109963299
Permalink: http://rave.ohiolink.edu/etdc/view?acc_num=osu1109963299

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