Synergistic Interaction between Yeast Nucleotide Excision Repair Factors NEF2 and NEF4 in the Binding of Ultraviolet-damaged DNA

Abstract

Saccharomyces cerevisiae RAD4, RAD7, RAD16, and RAD23 genes function in the nucleotide excision repair (NER) of ultraviolet light (UV)-damaged DNA. Previous biochemical studies have shown that the Rad4 and Rad23 proteins are associated in a stoichiometric complex named NEF2, and the Rad7 and Rad16 proteins form another stoichiometric complex named NEF4. While NEF2 is indispensable for the incision of UV-damaged DNA in the in vitro reconstituted system, NEF4 stimulates the incision reaction. Both NEF2 and NEF4 bind UV-damaged DNA, which raises the intriguing possibility that these two complexes cooperate to achieve the high degree of specificity for DNA damage demarcation required for nucleotide excision repair in vivo. Consistent with this hypothesis, we find that NEF2 and NEF4 bind in a synergistic fashion to UV-damaged DNA in a reaction that is dependent on ATP. We also purify the Rad7 protein and show that it binds DNA but has no preference for UV-damaged DNA. Rad7 physically interacts with NEF2, suggesting a role for Rad7 in linking NEF2 with NEF4.In eukaryotes, nucleotide excision repair (NER) 1 of ultraviolet (UV)-damaged DNA is a highly intricate process which requires a large number of proteins. A defect in NER in yeast results in an extreme sensitivity to UV light and a hypermutational phenotype, and in humans, defective NER is the underlying cause of the skin cancer-prone syndrome xeroderma pigmentosum, observations that underscore the importance of this repair system in neutralizing the cytotoxicity and genotoxicity of UV light (1).During NER, the DNA lesion is first bound by damage recognition proteins, followed by the recruitment of additional NER factors and DNA unwinding by the two DNA helicases Rad3 and Rad25, present in TFIIH, to create a DNA "bubble." Dual incision of the damage-containing strand in the unwound DNA, on the 5Ј-side by the Rad1-Rad10 nuclease and on the 3Ј-side by Rad2, results in the release of the lesion in the form of an oligonucleotide ϳ 30 nucleotides in length (for a discussion, see Ref. 2). A conserved set of proteins performs the same function in humans (3-5). Biochemical fractionation of Saccharomyces cerevisiae extract has revealed distinct subassemblies of the NER proteins, termed nucleotide excision repair factors or NEFs. Thus, Rad14 associates with the Rad1-Rad10 endonuclease to form NEF1 (6), Rad4 and Rad23 combine to form NEF2 (7), and Rad2 associates with the six subunit RNA polymerase II transcription factor TFIIH to form NEF3 (2). The combination of NEF1, NEF2, NEF3, and the heterotrimeric ssDNA binding factor replication protein A (RPA) is sufficient to mediate dual incision of UV-damaged DNA in an in vitro reconstituted system (7), indicating that the basal NER machinery consists of these factors and also lends credence to the suggestion that NER is mediated by the stepwise incorporation of the aforementioned NEFs and RPA at the damage site (2).At the genomic level, another level of complexity exists in the repair of transcribed versus nontranscribed DNA. The...

DNA Repair Mechanisms and the Bypass of DNA Damage in Saccharomyces cerevisiae

“…Rad16 facilitates histone H3 acetylation after UV irradiation , however, and an effect of histone H3 methylation on NER at silenced loci has been reported (Chaudhuri et al 2009). Although not required for dual cleavage of UV-irradiated DNA in vitro, the Rad7-Rad16 complex markedly stimulates the reaction; combining Rad4-Rad23 and Rad7-Rad16 synergistically enhances binding to UV-damaged DNA (Guzder et al 1999). Finally, physical interaction between Rad4 and Rad7 allows the formation of a large lesion-recognition complex (Guzder et al 1999).…”

“…Although not required for dual cleavage of UV-irradiated DNA in vitro, the Rad7-Rad16 complex markedly stimulates the reaction; combining Rad4-Rad23 and Rad7-Rad16 synergistically enhances binding to UV-damaged DNA (Guzder et al 1999). Finally, physical interaction between Rad4 and Rad7 allows the formation of a large lesion-recognition complex (Guzder et al 1999). These data clearly point to a role for Rad7-Rad16 in DNA damage recognition during GG-NER, and other studies suggest an additional role for Rad7-Rad16 in post-incision events ).…”

The 19S complex of the proteasome regulates nucleotide excision repair in yeast

“…No catalytic function has been associated with yeast Rad23 protein. The protein forms a tight complex with Rad4 protein (a protein with limited amino-acid sequence homology to human XPC protein) through its carboxy-terminal region (Guzder et al 1995;Wang et al 1997;Schauber et al 1998) and the Rad23/Rad4 complex has been shown to preferentially bind UV-irradiated DNA (Guzder et al 1998(Guzder et al , 1999Jansen et al 1998). The amino-terminal region of Rad23 protein contains a ubl domain that is required for optimal levels of NER in vivo.…”

Evidence for the Involvement of Nucleotide Excision Repair in the Removal of Abasic Sites in Yeast

“…NER is a highly conserved process among eukaryotes from yeast to humans (36). In S. cerevisiae, NER is accomplished via the concerted action of the following: Rad14, RPA, the Rad4-Rad23 complex, and the Rad7-Rad16 complex, all of which function in DNA damage recognition (5,11,12,(14)(15)(16)22); TFIIH, which contains the Rad3 and Rad25 DNA helicases, essential for DNA unwinding (41); and the Rad1-Rad10 and Rad2 nucleases (18, 42, 43) which incise the damaged strand on the 5Ј and 3Ј sides of the lesion, respectively (2, 17). Both in yeast and humans, dual incision by the NER ensemble results in the release of an oligonucleotide fragment ϳ25 to 30 nucleotides long (10,28,29).…”