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• W1969270834 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. 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. nucleotide excision repair replication protein A ubiquinone nitrilotriacetic acid glutathione S-transferase adenosine 5′-O-(3-thiotriphosphate) 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 (1Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. ASM Press, Washington, D. C.1995Google Scholar). 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 5′-side by the Rad1-Rad10 nuclease and on the 3′-side by Rad2, results in the release of the lesion in the form of an oligonucleotide ∼ 30 nucleotides in length (for a discussion, see Ref. 2Habraken Y. Sung P. Prakash S. Prakash L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10718-10722Crossref PubMed Scopus (45) Google Scholar). A conserved set of proteins performs the same function in humans (3Mu D. Park C.-H. Matsunaga T. Hsu D.S. Reardon J.T. Sancar A. J. Biol. Chem. 1995; 270: 2415-2418Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar, 4Mu D. Hsu D.S. Sancar A. J. Biol. Chem. 1996; 271: 8285-8294Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 5Mu D. Wakasugi M. Hsu D.S. Sancar A. J. Biol. Chem. 1997; 272: 28971-28979Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). 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 (6Guzder S.N. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1996; 271: 8903-8910Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), Rad4 and Rad23 combine to form NEF2 (7Guzder S.N. Habraken Y. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1995; 270: 12973-12976Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar), and Rad2 associates with the six subunit RNA polymerase II transcription factor TFIIH to form NEF3 (2Habraken Y. Sung P. Prakash S. Prakash L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10718-10722Crossref PubMed Scopus (45) Google Scholar). 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 (7Guzder S.N. Habraken Y. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1995; 270: 12973-12976Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar), 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 (2Habraken Y. Sung P. Prakash S. Prakash L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10718-10722Crossref PubMed Scopus (45) Google Scholar). At the genomic level, another level of complexity exists in the repair of transcribed versus nontranscribed DNA. The repair of the transcribed strand requires the RAD26 gene, a homolog of the human CSB gene (8van Gool A.J. Verhage R. Swagemakers S.M.A. van de Putte P. Brouwer J. Troelstra C. Bootsma D. Hoeijmakers J.H.J. EMBO J. 1994; 13: 5361-5369Crossref PubMed Scopus (218) Google Scholar), whereas the repair of the nontranscribed strand requires the RAD7 and RAD16 genes (9Verhage R. Zeeman A.-M. de Groot N. Gleig F. Gang D.D. van de Putte P. Brouwer J. Mol. Cell. Biol. 1994; 14: 6135-6142Crossref PubMed Scopus (173) Google Scholar, 10Mueller J.P. Smerdon M.J. Nucleic Acids Res. 1995; 23: 3457-3464Crossref PubMed Scopus (28) Google Scholar). Deletion of RAD7 or RAD16 causes an intermediate level of UV sensitivity, and the UV sensitivity of the rad7Δ rad16Δ double mutant is the same as that of the rad7Δ and rad16Δ single mutants. The Rad7 and Rad16 proteins exist together in a stable complex termed NEF4, and the purified Rad7-Rad16 heterodimer binds UV-damaged DNA in an ATP-dependent manner (11Guzder S.N. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1997; 272: 21665-21668Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). NEF4 also has a DNA-dependent ATPase activity, and UV irradiation of DNA results in a marked inhibition of ATP hydrolysis (12Guzder S.N. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1998; 273: 6292-6296Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). These observations have suggested a model in which NEF4 utilizes the free energy from ATP hydrolysis to translocate on DNA. Because of the attenuation of the ATPase activity, NEF4 stops translocating at the damage site and becomes stably bound to the damage. In this scenario, NEF4 would be the first protein complex to arrive at the damage site and would serve as the nucleation site for the subsequent assembly of the other repair factors (11Guzder S.N. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1997; 272: 21665-21668Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 12Guzder S.N. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1998; 273: 6292-6296Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Consistent with this hypothesis, the addition of NEF4 to the reconstituted NER system results in a marked stimulation of the proficiency of the incision reaction (11Guzder S.N. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1997; 272: 21665-21668Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). In addition to NEF4, the Rad4-Rad23 complex (NEF2) also recognizes UV damage (13Guzder S.N. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1998; 273: 31541-31546Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 14Jansen L.E.T. Verhage R.A. Brouwer J. J. Biol. Chem. 1998; 273: 33111-33114Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). However, by contrast to the damage binding activity of NEF4, which is strongly dependent upon ATP, NEF2 damage binding shows no such dependence on a nucleotide co-factor (13Guzder S.N. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1998; 273: 31541-31546Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). To begin delineating the intricacy of the damage recognition step in NER, we examine here whether the combination of NEF2 and NEF4 is more adept at binding UV-damaged DNA than either protein complex alone. Our results indicate a synergistic enhancement of damage binding by NEF2 and NEF4. We also purify the Rad7 protein and show that it binds DNA but has no specificity for UV-damaged DNA. Furthermore, we provide evidence for the physical interaction of Rad7 with NEF2, which suggests a specific function for Rad7 as a bridging factor between NEF2 and NEF4. The anti-Rad7, anti-Rad4, anti-Rad16, and anti-Rad23 antibodies used in this study were all affinity purified from rabbit antisera raised against portions of the respective proteins expressed in and purified from Escherichia coli as described (11Guzder S.N. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1997; 272: 21665-21668Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 13Guzder S.N. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1998; 273: 31541-31546Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 15Watkins J.F. Sung P. Prakash L. Prakash S. Mol. Cell. Biol. 1993; 13: 7757-7765Crossref PubMed Scopus (217) Google Scholar). The construction of the Rad7 overexpression plasmid pR7.8 has been described previously (11Guzder S.N. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1997; 272: 21665-21668Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). For overexpression of the glutathione S-transferase (GST)-Rad7 fusion protein, a 2.3-kilobase Xba I-Eco RI fragment containing the entire Rad7 coding sequence was cloned in plasmid pGEX-3X to yield the GST-Rad7 expression plasmid pR7.4. The protease-deficient yeast strain LY2 harboring plasmid pR7.8 was grown overnight to the stationary phase in complete synthetic medium lacking leucine. The overnight starter culture was diluted ten times with fresh medium that also contained 2% galactose and was then incubated at 30 °C for 10 h before the cells were harvested by centrifugation. We obtained 300 g of yeast paste from 80 liters of culture. Cell paste (300 g) of yeast strain LY2 containing pR7.8 was resuspended in cell breakage buffer containing protease inhibitors and disrupted by the French press. After clarification of the crude cell lysate by centrifugation (100,000 × g, 90 min), the Rad7 protein was quantitatively precipitated by the addition of 0.22 g of ammonium sulfate per ml of lysate. The ammonium sulfate pellet was collected by centrifugation, and proteins were redissolved in K buffer (20 mm KH2PO4, pH 7.5, 10% glycerol, 0.5 mm EDTA, 1 mm dithiothreitol) to give Fraction I with an ionic strength equivalent to 100 mmKCl and then loaded onto a Q-Sepharose column (2.5 × 5 cm; 25 ml). The Q-Sepharose flow-through (Fraction II) was applied onto a SP-Sepharose column (2.5 × 5 cm; 25 ml), which was developed with a 250-ml gradient of 100–500 mm KCl in K buffer. Fractions containing the peak of Rad7 protein (Fraction III) eluting at an ionic strength of 220 mm KCl were pooled, loaded onto a MacroHAP hydroxyapatite column (Bio-Rad; 2 ml), and equilibrated in K buffer with 100 mm KCl, which was developed with a 40-ml gradient of 20–300 mm potassium phosphate in the same buffer. Fractions containing Rad7 (Fraction IV; 7 ml) eluting at an ionic strength equivalent to 150 mm phosphate were dialyzed against 1 liter of buffer A (20 mm Tris-Cl, pH 7.0, 10% glycerol, 0.5 mm EDTA, 0.01% Nonidet P-40, 250 mm KOAc, 1 mm 2-mercaptoethanol) for 5 h at 4 °C. The dialysate was mixed gently with 0.3 ml of Ni-NTA-agarose matrix (Qiagen) for 3 h at 4 °C. The matrix was washed successively with buffer containing 10, 20, 30, 40, and 100 mm imidazole. Most (>85%) of the Rad7 protein was recovered in the 100 mm imidazole eluate (Fraction V), which was loaded onto a Mono-S column (HR 5/5) equilibrated in buffer A containing 100 mm KCl. The Mono-S column was developed with a 12-ml gradient of 200–600 mm KCl, collecting 0.5-ml fractions. The fractions containing nearly homogeneous Rad7 protein were pooled (Fraction VI) and concentrated to a small volume using a Centricon 30 concentrator and stored in small aliquots at −70 °C. To facilitate purification of Rad23 protein from yeast cells, the RAD23 gene was cloned downstream of the highly expressed yeast ADC1 promoter to yield the multicopy plasmid pJW112 (2μm, ADC1-RAD23). Rad23 protein overexpressed in the protease-deficient yeast strain LP2749–9B was purified to homogeneity as described previously (16Guzder S.N. Bailly V. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1995; 270: 8385-8388Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The NEF2 complex, comprised of Rad4 and Rad23 proteins, was purified to homogeneity from yeast cells overexpressing the Rad4 protein alone because Rad23 protein is present in considerable molar excess over Rad4 protein. During the purification procedure, the Rad4 and Rad23 proteins were monitored by Western analysis using affinity purified antibodies as described previously (13Guzder S.N. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1998; 273: 31541-31546Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). The Rad14 protein, Rad1-Rad10 complex, TFIIH, and RPA were purified as described (7Guzder S.N. Habraken Y. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1995; 270: 12973-12976Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). A 130-base pair DNA fragment containing a stretch of thymine residues (17Guzder S.N. Sung P. Prakash L. Prakash S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5433-5437Crossref PubMed Scopus (88) Google Scholar) was labeled at the 5′-end with 32P and incubated with the indicated amounts of Rad7, NEF2, and NEF4 in 10 μl of reaction buffer (30 mmpotassium HEPES, pH 7.5, 30 mm KCl, 5 mmMgCl2, 1 mm dithiothreitol, 100 μg/ml bovine serum albumin) and containing either 50 ng or the indicated amounts of Hae III linearized ΦX174 double-stranded DNA as competitor DNA. After 10 min at 30 °C, samples were mixed with 2 μl of gel loading buffer (0.1 m Tris-HCl, pH 7.0, 50% glycerol, and 0.05% Orange G) and electrophoresed in a 4.5% polyacrylamide gel in TAE buffer (40 mm Tris acetate, pH 7.4, 1 mmEDTA) at 4 °C. The gels were dried and subjected to autoradiography using Kodak MR films, and autoradiograms were quantitated by a Bio-Rad GS670 densitometer. The NER reaction was carried out as described (7Guzder S.N. Habraken Y. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1995; 270: 12973-12976Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Briefly, 75 ng of NEF2, 50 ng of RPA, 65 ng of NEF1, 65 ng of NEF3, and 60 ng of NEF4 or Rad7, where indicated, were incubated in reaction buffer (45 mm potassium HEPES, pH 7.9, 8 mm MgCl2, 120 μg/ml bovine serum albumin, 1.5 mm dithiothreitol, 2 mm ATP, an ATP regenerating system consisting of 30 mm creatine phosphate and 200 ng of creatine kinase) with 150 ng of M13mp18 DNA that had been irradiated with UV light emitting at 254 nm to a total dose of 30 J/m2. Reaction mixtures were incubated at 30 °C for varying times, deproteinized by treatment with SDS and proteinase K (7Guzder S.N. Habraken Y. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1995; 270: 12973-12976Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar), and analyzed in a 0.8% agarose gel in TAE buffer (20 mm Tris acetate, pH 7.4, 0.5 mm EDTA). The DNA species were visualized by staining with ethidium bromide. The GST-Rad7 fusion protein was overexpressed in the E. coli strain JM101, using the plasmid pR7.4. After induction with 1 mm isopropyl-1-thio-b-d-galactopyranoside, cells were broken and the GST-Rad7 fusion protein, which was localized in the inclusion bodies, was solubilized in 1.5% Sarkosyl buffer containing 0.1 m ethanolamine and 10 mm EDTA (pH 8.0). Triton X-100 was added to 2% to the supernatant containing the GST-Rad7 protein, and after clarification, the preparation was dialyzed extensively against phosphate-buffered saline at 4 °C. The GST-Rad7 protein in this fraction was present at approximately 1.5 mg/ml. A Rad7 affinity matrix was generated by binding GST-Rad7 protein to glutathione-Sepharose beads according to the instructions of the manufacturer (Amersham Pharmacia Biotech). The matrix was washed with 10 volumes of buffer (20 mm Tris acetate, 20% glycerol, 0.5 mm EDTA, and 0.01% Nonidet P-40) containing 50 mm KOAc, pH 7.0 and then resuspended in an equal volume of buffer containing 50 mm KOAc. An aliquot (2.5 μl) of the beads was incubated with buffer containing 20 mm glutathione to check the amount of Rad7 bound to the matrix (1 μg/μl beads). A similar protocol was followed to generate GST-Sepharose beads, using lysate containing GST (6.4 μg/μl of beads). For binding experiments, purified Rad23 (0.5 μg) or NEF2 (1.5 μg) was mixed with 10 μl of the GST- or GST-Rad7 affinity matrix in 150 μl of buffer Q (20 mm Tris acetate, pH 7.0, 20% glycerol, 1.5 mmEDTA, 1 mm dithiothreitol, 0.01% Nonidet P-40) containing 50 mm KOAc at 4 °C for 3 h. After removal of the unbound proteins, the matrix was washed with 0.2 ml of buffer Q containing 0.2, 0.4, and 0.8 m KOAc. Proteins were then eluted into 40 μl of 2% SDS by incubation of the affinity beads containing bound proteins at 37 °C for 10 min. The supernatants (5 μl), washes (5 μl), and SDS-eluates (1.5 μl) were analyzed for the presence of NEF2 or Rad23 protein by immunoblotting. Binding of NEF4 to UV-damaged DNA is strongly stimulated by ATP or ATPγS (11Guzder S.N. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1997; 272: 21665-21668Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar,12Guzder S.N. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1998; 273: 6292-6296Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), whereas NEF2 shows no dependence on a nucleotide in its binding to UV-irradiated DNA (13Guzder S.N. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1998; 273: 31541-31546Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). To determine whether NEF2 and NEF4 interact synergistically in the binding of UV-damaged DNA, we used concentrations of NEF2 and NEF4 at which little binding of the UV-irradiated DNA fragment is seen with either factor alone and then investigated the effect of mixing the two NEFs in the presence of ATP. As shown in Fig. 1, A and B, whereas only ∼5% or less of the UV-damaged DNA fragment was bound by NEF2 or NEF4, combining the two NEFs resulted in a marked elevation in the level of binding (∼70%), an increase much higher than the simple sum of the binding activities seen with the two protein complexes (Fig. 1 B). Concomitant with the increased binding of the UV-damaged DNA fragment, nucleoprotein complexes that have retarded mobilities (marked collectively as C* in Fig. 1 A) compared with those formed between the DNA substrate and NEF2 or NEF4 alone (labeled C in Fig. 1 A) were seen. Immunoblotting after transfer of the nucleoprotein complex from the polyacrylamide gel onto nitrocellulose has indicated the presence of both NEF2 and NEF4 in the C* complex, thus confirming that C* was a ternary complex consisting of the damaged DNA, NEF2, and NEF4. Under the conditions used, undamaged DNA is not bound by the combination of NEF2 and NEF4 (Fig.1 C). Because NEF4 binds and hydrolyzes ATP, and the presence of either ATP or ATPγS is critical for optimal damage-specific DNA binding by NEF4 (11Guzder S.N. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1997; 272: 21665-21668Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 12Guzder S.N. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1998; 273: 6292-6296Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), it was of considerable interest to examine whether the synergistic cooperation between NEF2 and NEF4 in damage-specific DNA binding is dependent on the presence of ATP. To do this, NEF2 alone, NEF4 alone, and the combination of NEF2 and NEF4 were incubated with undamaged DNA or with UV-irradiated DNA in the absence or the presence of ATP. As shown in Fig. 1, C and D, combining NEF2 and NEF4 results in a synergistic increase in the binding of UV-damaged DNA. Significantly, the formation of the NEF2-NEF4-UV-damaged DNA ternary complex was greatly diminished when ATP was omitted from the reaction mixture (Fig. 1 C, compare lanes 16 and 17), indicating a requirement for ATP in the formation of the ternary nucleoprotein complex. As reported previously (11Guzder S.N. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1997; 272: 21665-21668Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 12Guzder S.N. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1998; 273: 6292-6296Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) and reiterated here, ATPγS, like ATP, was effective in promoting DNA damage binding by NEF4 (Fig. 1 C, compare lanes 14 and 15 with lane 13). ATPγS, however, did not stimulate the formation of the ternary complex of damaged DNA with NEF2 and NEF4 (Fig. 1 C, compare lanes 16 and 18). This result stands in contrast to the damage binding by NEF4 alone, and it suggests that either only ATP is effective in inducing a conformation conducive for the interaction of NEF4 with NEF2 or that ATP hydrolysis is in fact indispensable for mediating the interaction of NEF2 with NEF4. No significant binding of the undamaged DNA fragment was seen even with the combination of NEF2 and NEF4, regardless of whether a nucleotide was present or not (Fig.1 C). The Rad7 protein does not contain any known conserved sequence motifs that might suggest a biochemical function for the protein. To gain insight into the biochemical role of Rad7, we overexpressed the protein in yeast cells (Fig. 2 A) and purified it to near homogeneity (Fig. 2 B). For the overexpression of Rad7 protein, the RAD7 gene tagged with a 6-histidine sequence at the amino terminus was placed under the control of the GAL-PGK promoter to yield plasmid pR7.8. For purifying Rad7 protein, extract from 300 g of the protease-deficient yeast strain LY2 harboring plasmid pR7.8 was subjected to ammonium sulfate precipitation, followed by chromatographic fractionation in columns of Q-Sepharose, SP-Sepharose, hydroxyapatite, nickel NTA-agarose, and finally in Mono-S, as described under “Materials and Methods.” The elution of Rad7 from the various columns was monitored by immunoblotting until the hydroxyapatite step, where Rad7 could be identified by Coomassie Blue staining of polyacrylamide gels in which the column fractions had been run. When 1 μg of Rad7 protein eluting from the last step of purification in Mono-S column (Fraction VI Rad7 protein) was analyzed by denaturing polyacrylamide gel electrophoresis and Coomassie Blue staining, only the Rad7 protein was seen, indicating a high degree of purity of the preparation (Fig. 2 B). The purified Rad7 preparation was devoid of Rad16 protein, as no Rad16 was detected when 1 μg of the Rad7 preparation was subjected to immunoblot analysis with anti-Rad16 antibodies under conditions wherein even 1 ng of Rad16 would have been seen. We obtained 60 μg of Rad7 protein from the starting 300 g of yeast paste used, representing an overall recovery of 10%. Fraction VI Rad7 protein was used in the DNA binding and in vitro repair reactions described below. Because NEF4 has a UV damage-specific DNA binding activity, we examined whether Rad7 protein alone can bind UV-damaged DNA. The results in Fig.3 A show that Rad7 has a DNA binding activity as indicated by the formation of a nucleoprotein complex with the 32P-labeled DNA fragment. However, unlike the Rad7-Rad16 complex, Rad7 protein by itself does not discriminate between the UV-damaged and undamaged DNA, as essentially the same level of nucleoprotein complex was formed with both DNA species (Fig.3 A). The same results were obtained when ATP was omitted from the reaction buffer (data not shown). Furthermore, the binding of Rad7 to either the UV-irradiated or unirradiated DNA substrate can be effectively competed away by adding low amounts of a nonlabeled DNA to the preformed nucleoprotein complex (Fig. 3 A), suggesting that the Rad7-DNA nucleoprotein complex is not very stable. In sharp contrast, NEF4 binds specifically and stably to UV-irradiated DNA even in the presence of a 100-fold excess of competitor DNA (Fig.3 B, lane 7). These results indicate that Rad7 is a DNA binding protein but it is devoid of the ability to recognize UV lesions. In further characterizing the function of Rad7 protein, we considered the possibility that Rad7 may act as a physical link between NEF2 and NEF4. Because even with overexpression, only a small amount of Rad7 protein could be purified from yeast cells, to facilitate our study, we expressed Rad7 as a fusion polypeptide to GST and purified it from E. coli according to standard procedure entailing affinity chromatography on glutathione-Sepharose. For examining whether there was a specific interaction between Rad7 and NEF2, either GST alone or purified GST-Rad7 immobilized on glutathione-Sepharose was incubated with purified NEF2. The GST-Rad7 affinity matrix and the control GST matrix were washed with increasing concentrations of potassium acetate, and the bound NEF2 was eluted by treatment with SDS. The starting material, the supernatant containing unbound NEF2, the potassium acetate washes, and the SDS eluates were subjected to immunoblotting with anti-Rad4 and anti-Rad23 antibodies to determine the amount of NEF2 that was specifically retained on the GST-Rad7 affinity matrix. Significantly, whereas greater than 90% of the input NEF2 was retained on the GST-Rad7 matrix, there was no binding of NEF2 to the GST matrix (Fig.4 A). Because we had purified Rad23 protein available (16Guzder S.N. Bailly V. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1995; 270: 8385-8388Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), we examined whether there was a direct interaction between Rad23 and GST-Rad7. However, under conditions wherein NEF2 interacts strongly with GST-Rad7, we did not observe any binding of the Rad23 protein to the GST-Rad7 matrix (Fig.4 B). Taken together, the results demonstrate a specific and direct interaction of Rad7 with NEF2, but the interaction may be through the Rad4 protein or it may occur only with the Rad4-Rad23 complex. Rad7 by itself shows no damage-specific binding (Fig. 3 A), and it did not enhance the binding of UV-damaged DNA by NEF2 (data not shown). In a previous study (11Guzder S.N. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1997; 272: 21665-21668Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), we showed that the addition of NEF4 to an in vitro NER reaction reconstituted with highly purified NEF1, NEF2, NEF3, and RPA markedly increased the efficiency of the damage-specific incision reaction. Because Rad7 interacts with NEF2, we examined whether the repair efficiency could be enhanced by the inclusion of the Rad7 protein in the reconstituted NER reaction. The addition of Rad7 to the basic incision reaction, however, did not result in significant stimulation or inhibition of the UV damage-specific incision (Fig.5 A, compare lanes 10 and 11 with lanes 6 and 7, respectively, and Fig. 5 B), whereas, as reported previously, the inclusion of NEF4 enhances the incision reaction by 4–5-fold (Fig.5 A, compare lanes 8 and 9 with lanes 6 and 7, respectively, and Fig.5 B). These biochemical results and the results from previous genetic studies are consistent with the suggestion that both Rad7 and Rad16 proteins are required for the functional integrity of NEF4 in UV damage recognition and in NER enhancement. Genetic studies have indicated that the RAD4 gene is indispensable for the NER of UV-damaged DNA in vivo, whereas the RAD23 gene affects the efficiency of the repair process (15Watkins J.F. Sung P. Prakash L. Prakash S. Mol. Cell. Biol. 1993; 13: 7757-7765Crossref PubMed Scopus (217) Google Scholar). Previous studies from our laboratory have shown that the Rad4 and Rad23 proteins are tightly associated in a stoichiometric complex called NEF2. Consistent with the genetic results, NEF2 was shown to be indispensable for the dual incision of UV-damaged DNA in the in vitro NER reconstitution studies (7Guzder S.N. Habraken Y. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1995; 270: 12973-12976Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Because the Rad4 and Rad23 proteins contain no identifiable sequence motifs that would predict an enzymatic activity, it had remained unclear as to whether NEF2 was important for NER by providing a distinct biochemical function or whether it primarily served as a scaffold in the assembly of the NER machinery. In fact, our previous studies have demonstrated that Rad23 interacts with Rad14 protein and with TFIIH (16Guzder S.N. Bailly V. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1995; 270: 8385-8388Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). More recently, we showed that NEF2 possesses a DNA binding ability that is highly specific for UV-damaged DNA, but Rad23 by itself shows no DNA binding ability (13Guzder S.N. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1998; 273: 31541-31546Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), indicating that either Rad4 is the damage recognition subunit of NEF2, or Rad4 and Rad23 proteins are both required for the DNA damage binding activity of NEF2. Similar to the DNA damage recognition ability of NEF2, the equivalent human NER complex consisting of the XPC and HHR23B proteins, also binds preferentially to UV-damaged DNA (18Reardon J.T. Mu D. Sancar A. J. Biol. Chem. 1996; 271: 19451-19456Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 19Sugasawa K. Ng J.M.Y. Masutani C. Iwai S. van der Spek P.J. Eker A.P.M. Hanaoka F. Bootsma D. Hoeijmakers J.H.J. Mol. Cell. 1998; 2: 223-232Abstract Full Text Full Text PDF PubMed Scopus (742) Google Scholar). Taken together, the biochemical studies have provided tangible evidence that during NER, NEF2 serves as a damage recognition factor, and in addition, plays an important role in the assembly of the incision machinery at the damage site via specific protein-protein interactions (16Guzder S.N. Bailly V. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1995; 270: 8385-8388Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The RAD7 and RAD16 genes affect the efficiency of NER in vivo with a particular requirement of these genes in the repair of nontranscribed DNA (9Verhage R. Zeeman A.-M. de Groot N. Gleig F. Gang D.D. van de Putte P. Brouwer J. Mol. Cell. Biol. 1994; 14: 6135-6142Crossref PubMed Scopus (173) Google Scholar, 10Mueller J.P. Smerdon M.J. Nucleic Acids Res. 1995; 23: 3457-3464Crossref PubMed Scopus (28) Google Scholar). The addition of Rad7-Rad16 complex, NEF4, to the reconstituted NER reaction results in a marked stimulation of the incision of UV-damaged DNA (11Guzder S.N. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1997; 272: 21665-21668Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Consistent with the presence in Rad16 of Walker-type sequence motifs suggestive of the ability to bind and hydrolyze a nucleoside triphosphate, our biochemical studies have indicated that NEF4 contains an intrinsic ATPase activity. The NEF4 ATPase activity requires DNA, and double-stranded DNA is more effective than single-stranded DNA for its activation (12Guzder S.N. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1998; 273: 6292-6296Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Like NEF2, NEF4 also binds specifically to UV-damaged DNA. However, unlike NEF2, which shows no dependence on ATP for damage binding, NEF4 requires ATP for maximal binding to UV-damaged DNA. ATPγS is also effective, but to a lesser degree than ATP, in promoting DNA damage binding by NEF4 (12Guzder S.N. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1998; 273: 6292-6296Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), which has led to the deduction that ATP binding alone is sufficient to induce a conformational change in NEF4 conducive for damage binding. The ATPase activity of NEF4 is markedly inhibited by the presence of UV damage in the DNA (12Guzder S.N. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1998; 273: 6292-6296Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), suggesting that the free energy derived from ATP hydrolysis may fuel the translocation of NEF4 on DNA and that the movement of NEF4 is arrested upon encountering a DNA lesion. The lesion-bound NEF4 may then serve as the nucleation site for the loading of the remaining NER factors, including NEF1, NEF2, NEF3, and RPA. In addition to NEF2, Rad14, which is a component of NEF1, and RPA also bind UV-damaged DNA specifically (17Guzder S.N. Sung P. Prakash L. Prakash S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5433-5437Crossref PubMed Scopus (88) Google Scholar, 20Clugston C.K. McLaughlin K. Kenny M.K. Brown R. Cancer Res. 1992; 52: 6375-6379PubMed Google Scholar, 21Burns J.L. Guzder S.N. Sung P. Prakash S. Prakash L. J. Biol. Chem. 1996; 271: 11607-11610Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar); however, the sequence by which these damage recognition factors assemble at the site of lesion-bound NEF4 remains to be determined. To begin addressing the hypothesis that multiple damage recognition factors including NEF2 and NEF4 function together to achieve a high degree of specificity in damage demarcation during NER, we have now examined NEF2 and NEF4 for their possible cooperation in DNA damage recognition. Our results indicate that combining NEF2 and NEF4 results in synergistic binding of UV-irradiated DNA. We also find that the synergistic action of NEF2 and NEF4 in damage recognition is an ATP-dependent process, suggesting a role of ATP binding/hydrolysis in this reaction. To explore the role of Rad7 in NER, we overexpressed and purified this protein to near homogeneity. Our results show that Rad7 binds DNA but has no specificity for UV-damaged DNA template. Consistent with this result, addition of NEF4, but not of Rad7 alone, to the in vitro NER reaction results in marked stimulation of the incision reaction (Fig. 5). These observations suggest that either Rad16 protein by itself provides damage-specific binding in NEF4 or that the combination of Rad7 and Rad16 proteins is in fact necessary for the expression of damage-specific DNA binding. These scenarios have not yet been tested directly, since despite considerable efforts to purify the Rad16 protein we have thus far been unable to obtain a sufficient amount of highly purified Rad16 for these studies, which is because of the extremely low level of expression of Rad16 protein in the absence of Rad7 protein. Nevertheless, the fact that Rad16 contains two potential zinc binding, DNA binding motifs, a C4 motif and a C3HC4 ring finger motif (22Prakash S. Sung P. Prakash L. Annu. Rev. Genet. 1993; 27: 33-70Crossref PubMed Scopus (257) Google Scholar), supports the suggestion that Rad16 by itself can discern between normal and damaged DNA species. If that was the case, then it is quite possible that the DNA binding ability of Rad7 has a role in enhancing the affinity of Rad16 for damaged DNA, or it increases the stability of the nucleoprotein complex formed between NEF4 and damaged DNA. In addition to the DNA binding activity, Rad7 also has the ability to physically interact with NEF2. Rad7 contains regions of marked hydrophobicity, including 12 tandemly repeated leucine-rich motifs (22Prakash S. Sung P. Prakash L. Annu. Rev. Genet. 1993; 27: 33-70Crossref PubMed Scopus (257) Google Scholar), which may enable Rad7 to interact with NEF2. In summary, our results suggest that NEF4 functions as an ATP-dependent DNA damage sensor and as an assembly factor during the incision phase of NER, and they provide support for the notion that the damage recognition reaction involves hierarchical interactions among different damage binding factors. Recent studies have suggested an involvement of the Rad7 and Rad16 proteins in the post-incision phase of NER (23Reed S.H. You Z. Friedberg E.C. J. Biol. Chem. 1998; 273: 29481-29488Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Thus, it appears that NEF4 plays a multifunctional role in NER, viz. in the very initial step of damage recognition in cooperation with NEF2 as shown in our studies here and also in the post-incision phase (23Reed S.H. You Z. Friedberg E.C. J. Biol. Chem. 1998; 273: 29481-29488Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), perhaps in the turnover of the incision protein machinery, and in the recruitment of factors to perform the DNA repair synthesis reaction." @default.
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