Biochemical and genetic analyses of nucleotide excision repair factor 4 in Saccharomyces cerevisiae

Nucleotide excision repair is the primary pathway for removal of UV-induced lesions in DNA. Nucleotide Excision Repair Factor 4 (NEF4) is required for repair of the non-transcribed strand of transcribed genes and transcriptionally silent DNA (Verhage et al. 1994; Bang et al. 1995). NEF4 was originally defined as the Rad16-Rad7 complex in yeast (Guzder et al. 1997), however I re-define NEF4 to include a novel subunit, Elc1, and demonstrate that Rad16, Rad7, and Elc1 co-associate (Chapter 3). Rad16 is a member of the Snf2/Swi2 ATPase family and contains a RING-H2 domain embedded within its ATPase domain (Bang et al. 1992; Mannhaupt et al. 1992; Eisen et al. 1995). Rad7 has previously unrecognized similarity to the F-box protein Grr1 and Elc1 is the yeast homolog of Elongin C, a mammalian E3 subunit. The presence of these motifs in NEF4 suggests that it functions as both an ATPase and as an E3 ubiquitin protein ligase. To test this hypothesis, a detailed analysis of the domains in Rad16 and Rad7 employed UV survival assays and immunoprecipitation experiments of Rad 16 and Rad7 mutants. My mutational analysis provides strong support for this model (Chapter 2). Furthermore, my genetic analysis uncovered new interactions between NEF4 and the NEF2 subunit Rad23, a repair factor that links nucleotide excision repair to proteasome function (Schauher et al. 1998). Rad23 is also known to control Rad4 levels (Lommel et al. 2002), which, together with data shown in Chapter 4, suggests that NEF4 is part of a complex system for globally regulating repair activity in vivo by controlling turnover of Rad4. Also presented in Chapter 4 are results from immunoprecipitation experiments to test for the direct interaction between Rad7 and previously reported NEF4-interacting proteins Abf1 and Sir3 (Paetkau et al. 1994; Reed et al. 1999). These results demonstrate that neither Abf1 nor Sir3 associate with Rad7. However, an affinity chromatography experiment identified multiple proteins that interact with Rad7 and Elc1, including Pdb1, Lat1, Snf4, Fpr4, and Rpa34. These interactions suggest that these newly-identified Rad7 and Elc1 interacting proteins might be potential substrates for NEF4's E3 activity or novel subunits of NEF4.