Date of Graduation

Summer 2011

Degree

Master of Science in Cell and Molecular Biology

Department

Biomedical Sciences

Committee Chair

Joshua Smith

Abstract

Nucleotide Excision Repair (NER) is a major DNA repair mechanism after UV light exposure. Rad23 in combination with Rad4, Nucleotide Excision Repair Factor 2 (NEF2), play a critical role in DNA damage recognition. Mutation or loss of the RAD4 human homolog, XPC, can lead to a serious disorder, Xeroderma Pigmentosum, which is characterized by increased sensitivity to sunlight and predisposition to cancer. Little is known about how Rad23 can function in both these roles and what signals are needed to stabilize a protein verses send it to the proteasome for degradation. Current research in Dr. Smith‟s lab has shown that the ubiquitin like (UBL) domain of Rad23 is often found to be ubiquitinated after UV light exposure which coincides with the increased level of Rad4. The objective of this project is to determine if the ubiquitination on Rad23 is involved in Rad4 stabilization in yeast, Saccharomyces. In order to determine the site of UV-dependent ubiquitin modification within the UBL domain of Rad23, 14 lysine residues were replaced by arginine individually as well as in various combinations. Molecular techniques such as UV sensitivity assays and western blots were applied to study the functional properties of these Rad23 mutants in Saccharomyces, and the role of ubiquitination following UV exposure. The site directed mutagenesis performed in this study has reiterated the importance of UBL domain since the combination mutants (lysine substituted by arginine) were UV sensitive.

Keywords

NER, Rad23, Rad4, XPC, UBL, ubiquitination, Saccharomyces, DNA damage

Subject Categories

Medical Molecular Biology

Copyright

© Archana none Shrestha

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