Publication Date

2011-06-07

Availability

Open access

Embargo Period

2011-06-07

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Biochemistry and Molecular Biology (Medicine)

Date of Defense

2011-06-03

First Committee Member

Feng Gong

Second Committee Member

Kenneth E. Rudd

Third Committee Member

Terace M. Fletcher

Fourth Committee Member

Antoni Barrientos

Abstract

DNA damage repair is an essential and complex cellular process. Although the basic mechanisms of nucleotide excision repair (NER) have been studied for decades, some mechanistic details remain elusive. The lesion detection step remains one of the most elusive in the process of NER in the contest of chromatin. The work described herein addresses the initial events in the lesion detection step of chromatin repair, also referred to as global genome repair (GG-NER). Both the role of post-translational modifications of lesion identification proteins, and the initial sequence of events in recruitment of repair and remodeling factors are investigated. First, the controversial role of ubiquitination of DDB2 (a human lesion detection protein) is investigated. Due to documented DDB2 function in alternative physiological processes, its direct role in GG-NER is hard to study in human cells. To overcome this obstacle, we established the budding yeast, Saccharomyces cerevisiae as an alternative, simplified model organism to study DDB2-mediated GG-NER. Using this system, we show that inconsistent with the widely accepted model, rapid degradation of DDB2 post-UV irradiation is not an absolute requirement for progression of GG-NER. However, interestingly, our data suggest a role for ubiquitination in the release of DDB2 from chromatin. In both UV and mock treated samples, ubiquitin deficient cells had significantly higher amounts of DDB2 remaining bound to the chromatin compared to the isogenic parent cells. The discussion focuses on the possible physiological relevance of these observations. Additionally, the recruitment of the SWI/SNF chromatin remodeling complex to the silent HML (Hidden MAT Left) locus was also investigated. SWI/SNF is known to require recruitment for its role in transcription; therefore we investigate this requirement in GG-NER. Based on previously published data that indicate an UV-stimulated association of SWI/SNF and Rad4 (a lesion detection protein), we hypothesized that Rad4 is involved in recruitment of SWI/SNF to damaged DNA. Interestingly, our data suggest that Rad4 is not an absolute requirement for recruitment of Snf6 to the HML locus following UV irradiation. However, Rad16 appears to be. These data present an interesting insight into the lesion detection step in GG-NER and this will be discussed.

Keywords

chromatin; DNA damage repair; Nucleotide excision repair (NER); DDB2; ubiquitination; Saccharomyces cerevisiae

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