Date of Award

1-1-2011

Document Type

Campus Access Dissertation

Department

College of Pharmacy

Sub-Department

Pharmaceutical Science

First Advisor

Douglas Pittman

Abstract

To preserve genome integrity, repair mechanisms must be in place to fix spontaneous and environmentally induced DNA damage. One of the most critical DNA repair pathways is homologous recombination (HR), which is responsible for error-free repair of DNA double-stranded breaks and interstrand crosslinks. Understanding the processes that regulate HR will provide new insights into carcinogenesis and potentially identify new targets for therapeutic intervention. The work presented in this dissertation investigated two novel mechanisms that govern the HR pathway, gene regulation by alternative mRNA splicing and post-translational modification.

The initial focus was on RAD51D, a central component of HR previously demonstrated to repair DNA damage as well as maintain chromosome and telomere stability. In the first study, mouse Rad51d alternative transcript products were found to make specific interactions with RAD51C and XRCC2. In addition, Rad51d alternative transcripts exhibited tissue-specific expression patterns in both mouse and human cells. This study suggested that RAD51D splice variants have the ability to sequester HR components into inactive complexes. Thus, differential expression of Rad51d alternative transcripts potentially regulates HR.

As part of a second study, proteins capable of post-translationally modifying the RAD51C, RAD51D, or XRCC2 HR proteins were discovered by a yeast two-hybrid screen. Because ubiquitin signaling proteins are emerging as essential regulators of the HR pathway, the follow-up work focused on an E3 ubiquitin ligase named RNF138. Interestingly, RNF138 specifically interacted with and stimulated RAD51D ubiquitination. Consistent with a role during HR, reduced RNF138 expression conferred heightened sensitivity to DNA damaging agents and increased levels of chromosome aberrations. This study also indicated that RNF138 dependent ubiquitination of RAD51D is an essential step for RAD51D degradation and for its role in HR DNA repair. In addition, RNF138 was found to have increased expression in breast and colon carcinoma cell lines, potentially resulting in reduction of RAD51D activity and contributing to genomic instability during carcinogenesis. These data imply that the RNF138 E3 ligase regulates RAD51D activity and may be a good therapeutic target to sensitize tumorigenic cells to DNA crosslinking agents.

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