Yeast Rad7-Rad16 Complex, Specific for the Nucleotide Excision Repair of the Nontranscribed DNA Strand, Is an ATP-dependent DNA Damage Sensor

Abstract

In eukaryotes, nucleotide excision repair of ultraviolet light-damaged DNA is a highly intricate process that requires a large number of evolutionarily conserved protein factors. Genetic studies in the yeast Saccharomyces cerevisiae have indicated a specific role of the RAD7 and RAD16 genes in the repair of transcriptionally inactive DNA. Here we show that the RAD7-and RAD16-encoded products exist as a complex of 1:1 stoichiometry, exhibiting an apparent dissociation constant (K d ) of <4 ؋ 10 ؊10 M. The Rad7-Rad16 complex has been purified to near homogeneity in this study and is shown to bind, in an ATP-dependent manner and with high specificity, to DNA damaged by ultraviolet light. Importantly, inclusion of the Rad7-Rad16 complex in the in vitro nucleotide excision repair system that consists entirely of purified components results in a marked stimulation of damage specific incision. Thus, Rad7-Rad16 complex is the ATP-dependent DNA damage sensor that specifically functions with the ensemble of nucleotide excision repair factor (NEF) 1, NEF2, NEF3, and replication protein A in the repair of transcriptionally inactive DNA. We name this novel complex of Rad7 and Rad16 proteins NEF4.In eukaryotes, nucleotide excision repair (NER) 1 of ultraviolet light-damaged DNA occurs by dual incision of the DNA strand that contains the UV lesion, excising the damage in the form of a short DNA fragment ϳ30 nucleotides in length (1). Mutational inactivation of NER in humans results in the cancer-prone syndrome xeroderma pigmentosum, which underscores the importance of this repair system in neutralizing the genotoxicity of UV light. The dual incision event in NER is accomplished by the concerted action of a large number of evolutionarily conserved proteins, and our studies in the yeast Saccharomyces cerevisiae have indicated an organization of these proteins into distinct subassemblies: nucleotide excision repair factor I, or NEF1, consisting of the damage recognition protein Rad14 and the Rad1-Rad10 endonuclease (2), NEF2, containing the Rad4 and Rad23 proteins (3), and NEF3, comprising the endonuclease Rad2 together with the RNA polymerase II transcription factor TFIIH (4). The combination of NEF1, NEF2, NEF3, and the heterotrimeric replication protein A (RPA) is sufficient for ATP-dependent dual incision to occur, indicating that the basic NER machinery is composed of these protein subassemblies (3). Dual incision of UV-damaged DNA can also be accomplished by combining the human equivalents of the aforementioned yeast repair proteins (5-7).At the genomic level, two functionally distinct modes of NER have been described, the first concerns with the repair of the transcribed strand in transcriptionally active chromosomal DNA, which involves the stalling of RNA polymerase II at the DNA lesion, and requires the CSA and CSB gene products (8). The second mode of excision repair is specific for the nontranscribed strand and for genomic regions that are transcriptionally inactive. Interestingly, in the rad7 and rad16 mutants of S. c...

Apurinic Endonuclease Activity of Yeast Apn2 Protein

“…Such a possibility is supported from studies of nucleotide excision repair in yeast, where the repair of UV damage from the nontranscribed regions of the genome is strictly dependent upon the RAD7 and RAD16 genes (15,16). In vitro, however, the incision reaction can be reconstituted on UV-damaged DNA in the absence of Rad7 and Rad16 proteins (17), although the addition of Rad7-Rad16 complex to the purified reconstituted system stimulates the incision reaction (18). Thus, the absolute need for the Rad7 and Rad16 proteins is manifested in vivo but not in vitro.…”

“…Thus, the absolute need for the Rad7 and Rad16 proteins is manifested in vivo but not in vitro. The Rad7-Rad16 protein complex has affinity for UV-damaged DNA, and it has been proposed that the Rad7-Rad16 complex functions in locating the damage in nontranscribed regions of DNA (18,19). Although the damage can be recognized and incised in vitro on naked DNA without the Rad7 and Rad16 proteins, in the absence of these proteins, the search and demarcation of DNA damage may become quite inefficient in vivo.…”

“…By contrast to nucleotide excision repair, which involves the concerted action of several distinct multiprotein complexes (17)(18)(19)(20)(21), base excision repair has been thought to rely upon the sequential action of several independent enzymatic activities. For example, following the recognition and removal of the damaged base by a DNA glycosylase, an AP endonuclease is thought to cleave at the AP site (2,3).…”

The NEF4 Complex Regulates Rad4 Levels and Utilizes Snf2/Swi2-Related ATPase Activity for Nucleotide Excision Repair

“…The enzymatic activities (e.g., ATPase and structure-specific nuclease) and DNA binding properties of many proteins have been extensively described (2,12). Multiple pairwise interactions between these components have also been detected (12,19,21), and based on these interactions, the Saccharomyces cerevisiae NER components can be subdivided into four nucleotide excision repair factors, NEF 1, 2, 3, and 4 (reviewed in reference 2).…”

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).…”