Function of NEF4 in Nucleotide Excision Repair

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DESCRIPTION (provided by applicant): One of the main pathways for repair of damaged DNA is through Nucleotide Excision Repair (NER). Defects in NER in humans have linked the repair of damaged DNA by NER to the predisposition to cancer. The NER reaction is conserved across species and we have exploited the advantages of S. cerevisiae to define the role of the NER complex NEF4. NEF4 is required for the efficient repair of nontranscribed DNA in yeast. The NEF4 complex consists of three proteins: Rad16, Rad7, and Elc1. Rad16 contains an Snf2/Swi2 ATPase domain perhaps thought to be involved in scanning the DNA for damage. More recently, we have identified NEF4 as containing conserved motifs similar to other E3 ubiquitin ligase complexes. These results link a novel ubiquitin-mediated pathway to NER and the regulation of Rad4, a protein involved in DNA damage recognition. Through the use of in vitro and in vivo NER assays, we will define the function of the NEF4 ATPase and ubiquitin ligase activities. Additionally, Chromatin immunoprecipitation (ChIP) assays will allow us to define the role of NEF4 in regulation and recruitment of other NER components to the sites of damaged DNA.