The Yeast RAD7 and RAD16 Genes Are Required for Postincision Events during Nucleotide Excision Repair

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...

Section: Discussionsupporting

confidence: 71%

“…Recent studies have suggested an involvement of the Rad7 and Rad16 proteins in the post-incision phase of NER (23).…”

Section: Discussionmentioning

confidence: 99%

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

Guzder

Sung

Prakash³

et al. 1999

“…The recent development of reconstituted systems using highly purified proteins has enabled analyses of the detailed reaction mechanism of NER (7)(8)(9)(10). In yeast, some accessory or regulatory factors such as Rad7⅐Rad16 complex (11)(12)(13), ABF1 (14), and MMS19 (15)(16) have been reported to help mediate NER. The existence of such factors in humans, however, has not been clearly demonstrated.…”

Section: Nucleotide Excision Repair (Ner)mentioning

confidence: 99%

Damaged DNA-binding Protein DDB Stimulates the Excision of Cyclobutane Pyrimidine Dimers in Vitro in Concert with XPA and Replication Protein A

Wakasugi

Shimizu

Morioka

et al. 2001

Human cells contain a protein that binds to UV-irradiated DNA with high affinity. This protein, damaged DNA-binding protein (DDB), is a heterodimer of two polypeptides, p127 and p48. Recent in vivo studies suggested that DDB is involved in global genome repair of cyclobutane pyrimidine dimers (CPDs), but the mechanism remains unclear. Here, we show that in vitro DDB directly stimulates the excision of CPDs but not (6 -4)photoproducts. The excision activity of cell-free extracts from Chinese hamster AA8 cell line that lacks DDB activity was increased 3-4-fold by recombinant DDB heterodimer but not p127 subunit alone. Moreover, the addition of XPA or XPA ؉ replication protein A (RPA), which themselves enhanced excision, also enhanced the excision in the presence of DDB. DDB was found to elevate the binding of XPA to damaged DNA and to make a complex with damaged DNA and XPA or XPA ؉ RPA as judged by both electrophoretic mobility shift assays and DNase I protection assays. These results suggest that DDB assists in the recognition of CPDs by core NER factors, possibly through the efficient recruitment of XPA or XPA⅐RPA, and thus stimulates the excision reaction of CPDs. Nucleotide excision repair (NER)1 is the major mechanism for removing bulky DNA lesions including cyclobutane pyrimidine dimers (CPDs) and (6 -4)photoproducts induced by sun light in humans (1-4). The basic reaction mechanism of NER is highly conserved from yeast to human. The core reaction from DNA damage recognition to excision of the damage is accomplished by six repair factors (XPA, RPA, XPC⅐HR23B, TFIIH, XPF⅐ERCC1, XPG), so called excision nuclease (excinuclease) (5-8). The recent development of reconstituted systems using highly purified proteins has enabled analyses of the detailed reaction mechanism of NER (7-10). In yeast, some accessory or regulatory factors such as Rad7⅐Rad16 complex (11-13), ABF1 (14), and MMS19 (15-16) have been reported to help mediate NER. The existence of such factors in humans, however, has not been clearly demonstrated.Damaged DNA-binding protein (DDB) is one of the candidates that might play such an accessory role in human cells. DDB is composed of two subunits, p127 and p48, and has a higher affinity for various types of DNA lesions compared with the damage recognition subunits (XPA, RPA, XPC⅐HR23B, and TFIIH) of the six core repair factors (17-19). DDB activity has been shown to be missing in a subset of xeroderma pigmentosum group E (XP-E) patients. XP is an autosomal recessive disease associated with abnormal sensitivity to UV lights and a high incidence of skin cancer, which is known to be caused by defects in NER. XP-E is one of eight genetic complementation groups (XP-A through XP-G and XP-V) of XP patients, and, along with XP-V, manifests the mildest clinical features and deficiency in NER. XP-E individuals lacking DDB activity were found to carry mutations in p48 gene (20 -23). Because of its high specificity for binding to damaged DNA and a defect in some XP-E cells, DDB has been postulated to be involved...

“…The Rad7 and Rad16 proteins also form a stable heterodimeric complex (16). Recent studies have demonstrated that these proteins are required for post-incision events during NER (15). Both proteins co-fractionate with recombinant Rad14 (Fig.…”

Section: His-tagged Rad14 Protein Complements Both the Uv Radiation Smentioning

confidence: 94%

“…2 Interestingly, extracts prepared from rad7, rad16, or rad23 strains, which exhibit moderate sensitivity to cell killing by UV radiation (5,6), are also defective in repair synthesis in vitro (14). Recent studies have shown that rad7 and rad16 mutant extracts are proficient in DNA damage-dependent incision both in vitro and in vivo but fail to excise the oligonucleotide fragment containing the DNA lesion (15). Hence, the Rad7 and Rad16 proteins, which interact with each other (16), appear to be required for post-incision events during NER.…”

mentioning

confidence: 99%

Affinity Purification and Partial Characterization of a Yeast Multiprotein Complex for Nucleotide Excision Repair Using Histidine-tagged Rad14 Protein

Rodriguez

Talamantez

Huang

et al. 1998

Self Cite

The nucleotide excision repair (NER) pathway of eukaryotes involves ϳ30 polypeptides. Reconstitution of this pathway with purified components is consistent with the sequential assembly of NER proteins at the DNA lesion. However, recent studies have suggested that NER proteins may be pre-assembled in a high molecular weight complex in the absence of DNA damage. To examine this model further, we have constructed a histidine-tagged version of the yeast DNA damage recognition protein Rad14. Affinity purification of this protein from yeast nuclear extracts resulted in the co-purification of Rad1, Rad7, Rad10, Rad16, Rad23, RPA, RPB1, and TFIIH proteins, whereas none of these proteins bound to the affinity resin in the absence of recombinant Rad14. Furthermore, many of the co-purifying proteins were present in approximately equimolar amounts. Co-elution of these proteins was also observed when the nuclear extract was fractionated by gel filtration, indicating that the NER proteins were associated in a complex with a molecular mass of >1000 kDa prior to affinity chromatography. The affinity purified NER complex catalyzed the incision of UV-irradiated DNA in an ATP-dependent reaction. We conclude that active high molecular weight complexes of NER proteins exist in undamaged yeast cells.