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W2041261463 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 RAD16genes 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 (Kd) of <4 × 10−10m. 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 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 RAD16genes 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 (Kd) of <4 × 10−10m. 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) 1The abbreviations used are: NER, nucleotide excision repair; NEF, nucleotide excision repair factor; RPA, replication protein A; ATPγS, adenosine 5′-O-(thiotriphosphate).1The abbreviations used are: NER, nucleotide excision repair; NEF, nucleotide excision repair factor; RPA, replication protein A; ATPγS, adenosine 5′-O-(thiotriphosphate). 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 ∼30 nucleotides in length (1Huang J.C. Svoboda D.L. Reardon J.T. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3664-3668Crossref PubMed Scopus (374) Google Scholar). 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 yeastSaccharomyces 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 (2Guzder S.N. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1996; 271: 8903-8910Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), NEF2, containing the Rad4 and Rad23 proteins (3Guzder S.N. Habraken Y. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1995; 270: 12973-12976Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar), and NEF3, comprising the endonuclease Rad2 together with the RNA polymerase II transcription factor TFIIH (4Habraken 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 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 (3Guzder S.N. Habraken Y. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1995; 270: 12973-12976Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). Dual incision of UV-damaged DNA can also be accomplished by combining the human equivalents of the aforementioned yeast repair proteins (5Mu 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, 6Mu D. Hsu D.S. Sancar A. J. Biol. Chem. 1996; 271: 8285-8294Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 7Moggs J.G. Yarema K.J. Essigmann J.M. Wood R.D. J. Biol. Chem. 1996; 271: 7177-7186Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar).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 theCSA and CSB gene products (8Hanawalt P.C. Science. 1994; 266: 1957-1960Crossref PubMed Scopus (452) Google Scholar). 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. cerevisiae, the nontranscribed strand is not repaired, while the repair of the transcribed strand is not affected (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), and transcription-independent NER is impaired in rad7 andrad16 mutant extracts (11He Z. Wong J.M.S. Maniar H.S. Brill S.J. Ingles C.J. J. Biol. Chem. 1996; 271: 28243-28249Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 12Wang Z. Wei S. Reed S.H. Wu X. Svejstrup J.Q. Feaver W.J. Kornberg R.D. Friedberg E.C. Mol. Cell. Biol. 1997; 17: 635-643Crossref PubMed Scopus (67) Google Scholar). Thus, the repair of the nontranscribed strand has a specific dependence on the RAD7and RAD16 genes.Here we describe our biochemical studies that help elucidate the molecular function of the Rad7 and Rad16 proteins in the repair of the nontranscribed strand. We show by co-immunoprecipitation that Rad7 and Rad16 proteins exist as a stable complex in yeast cells. The Rad7-Rad16 complex, which we have named NEF4, has been purified to near homogeneity from extract of a yeast strain co-expressing the two proteins. Using a DNA mobility shift assay, we demonstrate that NEF4 binds with high specificity and avidity to UV lesions and that this damage binding reaction has a strong dependence on ATP. Importantly, the addition of NEF4 to the reconstituted NER reaction results in a marked stimulation of damage-specific incision. Taken together, our results suggest that NEF4 is an ATP-dependent DNA damage sensor that functions specifically to target the basic NER machinery (viz. NEF1, NEF2, NEF3, and RPA) to the repair of nontranscribed DNA.RESULTS AND DISCUSSIONThe RAD7-encoded protein was overproduced in yeast by placing the RAD7 gene under the control of theGAL-PGK promoter-yielding plasmid pR7.8 (Fig. 1 A). To overproduce the Rad16 protein in yeast, the RAD16 gene was fused to the alcohol dehydrogenase I (ADC1) promoter, yielding plasmid pR16.15 (Fig. 1 A). When extract from the protease-deficient yeast strain LY2 co-harboring pR7.8 and pR16.15 was subjected to immunoprecipitation with protein A-agarose beads containing covalently conjugated anti-Rad7 and anti-Rad16 antibodies, co-precipitation of the Rad7 and Rad16 proteins was observed (Fig. 1 A, lanes 8 and9); no precipitation of either Rad7 or Rad16 protein was seen with beads containing antibodies specific for the Rad57 protein (Fig. 1 A, lane 7), which functions in recombinational repair (13Sung P. Genes Dev. 1997; 11: 1111-1121Crossref PubMed Scopus (459) Google Scholar). Control experiments showed that the precipitation of Rad7 by the anti-Rad16 immunobeads requires the presence of Rad16 protein and, likewise, that the precipitation of Rad16 protein by anti-Rad7 immunobeads is dependent upon Rad7 protein (Fig. 1 A). In immunoprecipitation experiments using extract from wild type yeast cells, co-precipitation of the Rad7 and Rad16 proteins was again observed. Thus, Rad7 and Rad16 proteins are complexed to each other in yeast cells.For purifying the Rad7-Rad16 protein complex, extract from LY2 co-overexpressing Rad7 and Rad16 proteins was subjected to the chromatographic fractionation scheme described under “Materials and Methods.” The Rad7 and Rad16 proteins remained quantitatively associated throughout all the purification steps, even in column fractions that contained as little as 4 × 10−10m of the protein complex, indicating a dissociation constant of the complex that is considerably lower than this protein concentration. When the Rad7-Rad16 protein complex from the last step of purification in Mono S (Fraction VI) was subjected to SDS-polyacrylamide gel electrophoresis and staining with Coomassie Blue, only the Rad7 and Rad16 bands were seen (Fig. 1, B andC), indicating a high degree of purity of the complex. Image analysis of the gel in Fig. 1 B revealed a one to one stoichiometry of the Rad7 and Rad16 proteins in the complex.We speculated that NEF4 might effect the repair of transcriptionally inactive DNA by sensing the DNA damage located in such regions and then recruiting the basic NER machinery to initiate the repair reaction. As a first test of this hypothesis, we examined whether NEF4 would bind UV-damaged DNA using a DNA mobility shift assay. The DNA fragment was labeled with 32P, irradiated with UV doses ranging from 1 to 12 kJ/m2, and then incubated with NEF4 in the presence of ATP and an excess of unlabeled φX174 DNA at 30 °C. After running the reaction mixtures in polyacrylamide gels under nondenaturing conditions, the gels were dried and exposed to x-ray films to reveal the radiolabeled DNA species. As shown in Fig. 2, incubation of the UV-irradiated DNA probe with NEF4 resulted in the formation of slower migrating forms of DNA, indicating binding of the damaged DNA by NEF4; no binding of NEF4 to the undamaged DNA was seen. The level of nucleoprotein complex formation was proportional to the amount of NEF4 (Fig. 2, Aand B) and to the UV dose (Fig. 2, C andD). Multiple nucleoprotein complexes were detected in these experiments, with the slower migrating nucleoprotein species being more prevalent at higher UV doses and with increasing concentrations of NEF4 (Fig. 2), suggesting that these species contained multiple NEF4 molecules bound to different damage sites in the DNA probe. Importantly, when ATP was omitted from the reaction, binding of the UV-damaged DNA decreased dramatically, such that only ∼6% of the UV-irradiated DNA was bound by NEF4 in the absence of the nucleotide, as compared with greater than 50% binding in its presence (Fig. 2 E). Although NEF4 possesses an ATPase activity that is activated by either single-stranded or double-stranded DNA, 2S. N. Guzder, unpublished observations. ATP binding by NEF4 is apparently sufficient for damage recognition, because the nonhydrolyzable ATP analogue ATPγS is nearly as effective as ATP in promoting damage binding (Fig. 2 E).Figure 2ATP-dependent binding of UV-damaged DNA by Rad7-Rad16 complex. A, protein concentration dependence of NEF4 binding to UV-damaged DNA. A 130-base pair DNA fragment with or without prior treatment with UV light (10 kJ/m2) was incubated with increasing amounts of NEF4, as indicated. The reaction mixture was resolved in a polyacrylamide gel, followed by autoradiography to visualize the nucleoprotein complexes (labeled collectively as C) and free DNA probe (labeled asF). B, graphical representation of the results inA •, undamaged DNA; ▴, UV damaged DNA. C, damage binding as a function of UV dose. NEF4, 60 ng, was incubated with a DNA fragment irradiated with increasing UV dose, as indicated.D, graphical representation of the results in C.E, nucleotide binding by NEF4 is sufficient for damage binding. NEF4, 60 ng, was incubated with a DNA fragment irradiated with 2 kJ/m2 in the absence of nucleotide (lane 2) and with 2 mm amounts of either ATPγS (lane 3) or ATP (lane 4). BL, DNA without NEF4 (lane 1).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Having established that NEF4 has high affinity for UV-damaged DNA, we then tested if NEF4 might improve the efficiency of the damage-specific incision reaction mediated by the basic NER machinery comprising NEF1, NEF2, NEF3, and RPA (2Guzder S.N. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1996; 271: 8903-8910Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 3Guzder S.N. Habraken Y. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1995; 270: 12973-12976Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 4Habraken Y. Sung P. Prakash S. Prakash L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10718-10722Crossref PubMed Scopus (45) Google Scholar, 15Sung P. Guzder S.N. Prakash L. Prakash S. J. Biol. Chem. 1996; 271: 10821-10826Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). To do this, we examined the rate of incision of supercoiled M13 DNA that had been exposed to 30 J/m2 UV light to introduce on average 1.8 photoproducts per plasmid DNA molecule (approximately 7.3 kilobase pairs long) by the basic NER machinery with or without added purified NEF4. Interestingly, addition of NEF4 to the in vitro NER reaction resulted in a marked stimulation of the conversion of the UV-irradiated supercoiled M13 plasmid DNA to the open circular form (Fig. 3, A and B), while no incision of the nondamaged M13 DNA was observed whether or not NEF4 was added to the repair reaction (Fig. 3 A). These observations indicate that NEF4 helps promote the damage-specific incision reaction. We also subjected the NER reaction to labeling with [α-32P]ddATP and calf thymus terminal transferase to detect the excision DNA fragments (3Guzder S.N. Habraken Y. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1995; 270: 12973-12976Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 15Sung P. Guzder S.N. Prakash L. Prakash S. J. Biol. Chem. 1996; 271: 10821-10826Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). In agreement with the results from the agarose gel method, the amount of excision fragments generated was also higher when NEF4 was included in the repair reaction (data not shown).Figure 3NEF4 promotes incision of UV-damaged DNA. A, M13mp18 DNA not treated (−UV) or treated with 30 J/m2 of UV (+UV) was incubated with NEF1, NEF2, and NEF3 (NEF1,2,3), with or without NEF4 at 30 °C for different times; RPA was added to all the repair reactions except the no protein controls in lanes 1 and 4. Reaction mixtures were run in a 0.8% agarose gel and stained with ethidium bromide to visualize the supercoiled (SC) and open circular DNA (OC), which was generated as a result of the damage-specific incision of the supercoiled form by the NER factors.B, graphical representation of the results in A. DNA incised corresponds to the percent of the supercoiled form that had been converted to the open circular form. Reaction mixtures with (•) or without NEF4 (▴) are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Our findings with NEF4 suggest that eukaryotes employ different mechanisms of damage recognition for the nontranscribedversus the transcribed strand. NEF4, an ATP-dependent damage recognition factor, is essential for the repair of the nontranscribed strand, whereas the products of theCSA and CSB/RAD26 genes are essential for preferential repair of the transcribed strand. NEF4 may be pivotal in locating the damage on the nontranscribed strand and in transcriptionally inactive regions of the genome, and it may do so by a tracking mechanism that utilizes the energy of ATP hydrolysis. Subsequent to binding the DNA lesion, NEF4 may serve as the nucleation site for the assembly of the other repair components for dual incision to occur. An additional role of NEF4 in the turnover of the incision enzyme complex is also possible. In eukaryotes, nucleotide excision repair (NER) 1The abbreviations used are: NER, nucleotide excision repair; NEF, nucleotide excision repair factor; RPA, replication protein A; ATPγS, adenosine 5′-O-(thiotriphosphate).1The abbreviations used are: NER, nucleotide excision repair; NEF, nucleotide excision repair factor; RPA, replication protein A; ATPγS, adenosine 5′-O-(thiotriphosphate). 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 ∼30 nucleotides in length (1Huang J.C. Svoboda D.L. Reardon J.T. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3664-3668Crossref PubMed Scopus (374) Google Scholar). 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 yeastSaccharomyces 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 (2Guzder S.N. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1996; 271: 8903-8910Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), NEF2, containing the Rad4 and Rad23 proteins (3Guzder S.N. Habraken Y. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1995; 270: 12973-12976Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar), and NEF3, comprising the endonuclease Rad2 together with the RNA polymerase II transcription factor TFIIH (4Habraken 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 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 (3Guzder S.N. Habraken Y. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1995; 270: 12973-12976Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). Dual incision of UV-damaged DNA can also be accomplished by combining the human equivalents of the aforementioned yeast repair proteins (5Mu 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, 6Mu D. Hsu D.S. Sancar A. J. Biol. Chem. 1996; 271: 8285-8294Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 7Moggs J.G. Yarema K.J. Essigmann J.M. Wood R.D. J. Biol. Chem. 1996; 271: 7177-7186Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). 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 theCSA and CSB gene products (8Hanawalt P.C. Science. 1994; 266: 1957-1960Crossref PubMed Scopus (452) Google Scholar). 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. cerevisiae, the nontranscribed strand is not repaired, while the repair of the transcribed strand is not affected (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), and transcription-independent NER is impaired in rad7 andrad16 mutant extracts (11He Z. Wong J.M.S. Maniar H.S. Brill S.J. Ingles C.J. J. Biol. Chem. 1996; 271: 28243-28249Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 12Wang Z. Wei S. Reed S.H. Wu X. Svejstrup J.Q. Feaver W.J. Kornberg R.D. Friedberg E.C. Mol. Cell. Biol. 1997; 17: 635-643Crossref PubMed Scopus (67) Google Scholar). Thus, the repair of the nontranscribed strand has a specific dependence on the RAD7and RAD16 genes. Here we describe our biochemical studies that help elucidate the molecular function of the Rad7 and Rad16 proteins in the repair of the nontranscribed strand. We show by co-immunoprecipitation that Rad7 and Rad16 proteins exist as a stable complex in yeast cells. The Rad7-Rad16 complex, which we have named NEF4, has been purified to near homogeneity from extract of a yeast strain co-expressing the two proteins. Using a DNA mobility shift assay, we demonstrate that NEF4 binds with high specificity and avidity to UV lesions and that this damage binding reaction has a strong dependence on ATP. Importantly, the addition of NEF4 to the reconstituted NER reaction results in a marked stimulation of damage-specific incision. Taken together, our results suggest that NEF4 is an ATP-dependent DNA damage sensor that functions specifically to target the basic NER machinery (viz. NEF1, NEF2, NEF3, and RPA) to the repair of nontranscribed DNA. RESULTS AND DISCUSSIONThe RAD7-encoded protein was overproduced in yeast by placing the RAD7 gene under the control of theGAL-PGK promoter-yielding plasmid pR7.8 (Fig. 1 A). To overproduce the Rad16 protein in yeast, the RAD16 gene was fused to the alcohol dehydrogenase I (ADC1) promoter, yielding plasmid pR16.15 (Fig. 1 A). When extract from the protease-deficient yeast strain LY2 co-harboring pR7.8 and pR16.15 was subjected to immunoprecipitation with protein A-agarose beads containing covalently conjugated anti-Rad7 and anti-Rad16 antibodies, co-precipitation of the Rad7 and Rad16 proteins was observed (Fig. 1 A, lanes 8 and9); no precipitation of either Rad7 or Rad16 protein was seen with beads containing antibodies specific for the Rad57 protein (Fig. 1 A, lane 7), which functions in recombinational repair (13Sung P. Genes Dev. 1997; 11: 1111-1121Crossref PubMed Scopus (459) Google Scholar). Control experiments showed that the precipitation of Rad7 by the anti-Rad16 immunobeads requires the presence of Rad16 protein and, likewise, that the precipitation of Rad16 protein by anti-Rad7 immunobeads is dependent upon Rad7 protein (Fig. 1 A). In immunoprecipitation experiments using extract from wild type yeast cells, co-precipitation of the Rad7 and Rad16 proteins was again observed. Thus, Rad7 and Rad16 proteins are complexed to each other in yeast cells.For purifying the Rad7-Rad16 protein complex, extract from LY2 co-overexpressing Rad7 and Rad16 proteins was subjected to the chromatographic fractionation scheme described under “Materials and Methods.” The Rad7 and Rad16 proteins remained quantitatively associated throughout all the purification steps, even in column fractions that contained as little as 4 × 10−10m of the protein complex, indicating a dissociation constant of the complex that is considerably lower than this protein concentration. When the Rad7-Rad16 protein complex from the last step of purification in Mono S (Fraction VI) was subjected to SDS-polyacrylamide gel electrophoresis and staining with Coomassie Blue, only the Rad7 and Rad16 bands were seen (Fig. 1, B andC), indicating a high degree of purity of the complex. Image analysis of the gel in Fig. 1 B revealed a one to one stoichiometry of the Rad7 and Rad16 proteins in the complex.We speculated that NEF4 might effect the repair of transcriptionally inactive DNA by sensing the DNA damage located in such regions and then recruiting the basic NER machinery to initiate the repair reaction. As a first test of this hypothesis, we examined whether NEF4 would bind UV-damaged DNA using a DNA mobility shift assay. The DNA fragment was labeled with 32P, irradiated with UV doses ranging from 1 to 12 kJ/m2, and then incubated with NEF4 in the presence of ATP and an excess of unlabeled φX174 DNA at 30 °C. After running the reaction mixtures in polyacrylamide gels under nondenaturing conditions, the gels were dried and exposed to x-ray films to reveal the radiolabeled DNA species. As shown in Fig. 2, incubation of the UV-irradiated DNA probe with NEF4 resulted in the formation of slower migrating forms of DNA, indicating binding of the damaged DNA by NEF4; no binding of NEF4 to the undamaged DNA was seen. The level of nucleoprotein complex formation was proportional to the amount of NEF4 (Fig. 2, Aand B) and to the UV dose (Fig. 2, C andD). Multiple nucleoprotein complexes were detected in these experiments, with the slower migrating nucleoprotein species being more prevalent at higher UV doses and with increasing concentrations of NEF4 (Fig. 2), suggesting that these species contained multiple NEF4 molecules bound to different damage sites in the DNA probe. Importantly, when ATP was omitted from the reaction, binding of the UV-damaged DNA decreased dramatically, such that only ∼6% of the UV-irradiated DNA was bound by NEF4 in the absence of the nucleotide, as compared with greater than 50% binding in its presence (Fig. 2 E). Although NEF4 possesses an ATPase activity that is activated by either single-stranded or double-stranded DNA, 2S. N. Guzder, unpublished observations. ATP binding by NEF4 is apparently sufficient for damage recognition, because the nonhydrolyzable ATP analogue ATPγS is nearly as effective as ATP in promoting damage binding (Fig. 2 E).Having established that NEF4 has high affinity for UV-damaged DNA, we then tested if NEF4 might improve the efficiency of the damage-specific incision reaction mediated by the basic NER machinery comprising NEF1, NEF2, NEF3, and RPA (2Guzder S.N. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1996; 271: 8903-8910Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 3Guzder S.N. Habraken Y. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1995; 270: 12973-12976Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 4Habraken Y. Sung P. Prakash S. Prakash L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10718-10722Crossref PubMed Scopus (45) Google Scholar, 15Sung P. Guzder S.N. Prakash L. Prakash S. J. Biol. Chem. 1996; 271: 10821-10826Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). To do this, we examined the rate of incision of supercoiled M13 DNA that had been exposed to 30 J/m2 UV light to introduce on average 1.8 photoproducts per plasmid DNA molecule (approximately 7.3 kilobase pairs long) by the basic NER machinery with or without added purified NEF4. Interestingly, addition of NEF4 to the in vitro NER reaction resulted in a marked stimulation of the conversion of the UV-irradiated supercoiled M13 plasmid DNA to the open circular form (Fig. 3, A and B), while no incision of the nondamaged M13 DNA was observed whether or not NEF4 was added to the repair reaction (Fig. 3 A). These observations indicate that NEF4 helps promote the damage-specific incision reaction. We also subjected the NER reaction to labeling with [α-32P]ddATP and calf thymus terminal transferase to detect the excision DNA fragments (3Guzder S.N. Habraken Y. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1995; 270: 12973-12976Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 15Sung P. Guzder S.N. Prakash L. Prakash S. J. Biol. Chem. 1996; 271: 10821-10826Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). In agreement with the results from the agarose gel method, the amount of excision fragments generated was also higher when NEF4 was included in the repair reaction (data not shown).Figure 3NEF4 promotes incision of UV-damaged DNA. A, M13mp18 DNA not treated (−UV) or treated with 30 J/m2 of UV (+UV) was incubated with NEF1, NEF2, and NEF3 (NEF1,2,3), with or without NEF4 at 30 °C for different times; RPA was added to all the repair reactions except the no protein controls in lanes 1 and 4. Reaction mixtures were run in a 0.8% agarose gel and stained with ethidium bromide to visualize the supercoiled (SC) and open circular DNA (OC), which was generated as a result of the damage-specific incision of the supercoiled form by the NER factors.B, graphical representation of the results in A. DNA incised corresponds to the percent of the supercoiled form that had been converted to the open circular form. Reaction mixtures with (•) or without NEF4 (▴) are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Our findings with NEF4 suggest that eukaryotes employ different mechanisms of damage recognition for the nontranscribedversus the transcribed strand. NEF4, an ATP-dependent damage recognition factor, is essential for the repair of the nontranscribed strand, whereas the products of theCSA and CSB/RAD26 genes are essential for preferential repair of the transcribed strand. NEF4 may be pivotal in locating the damage on the nontranscribed strand and in transcriptionally inactive regions of the genome, and it may do so by a tracking mechanism that utilizes the energy of ATP hydrolysis. Subsequent to binding the DNA lesion, NEF4 may serve as the nucleation site for the assembly of the other repair components for dual incision to occur. An additional role of NEF4 in the turnover of the incision enzyme complex is also possible. The RAD7-encoded protein was overproduced in yeast by placing the RAD7 gene under the control of theGAL-PGK promoter-yielding plasmid pR7.8 (Fig. 1 A). To overproduce the Rad16 protein in yeast, the RAD16 gene was fused to the alcohol dehydrogenase I (ADC1) promoter, yielding plasmid pR16.15 (Fig. 1 A). When extract from the protease-deficient yeast strain LY2 co-harboring pR7.8 and pR16.15 was subjected to immunoprecipitation with protein A-agarose beads containing covalently conjugated anti-Rad7 and anti-Rad16 antibodies, co-precipitation of the Rad7 and Rad16 proteins was observed (Fig. 1 A, lanes 8 and9); no precipitation of either Rad7 or Rad16 protein was seen with beads containing antibodies specific for the Rad57 protein (Fig. 1 A, lane 7), which functions in recombinational repair (13Sung P. Genes Dev. 1997; 11: 1111-1121Crossref PubMed Scopus (459) Google Scholar). Control experiments showed that the precipitation of Rad7 by the anti-Rad16 immunobeads requires the presence of Rad16 protein and, likewise, that the precipitation of Rad16 protein by anti-Rad7 immunobeads is dependent upon Rad7 protein (Fig. 1 A). In immunoprecipitation experiments using extract from wild type yeast cells, co-precipitation of the Rad7 and Rad16 proteins was again observed. Thus, Rad7 and Rad16 proteins are complexed to each other in yeast cells. For purifying the Rad7-Rad16 protein complex, extract from LY2 co-overexpressing Rad7 and Rad16 proteins was subjected to the chromatographic fractionation scheme described under “Materials and Methods.” The Rad7 and Rad16 proteins remained quantitatively associated throughout all the purification steps, even in column fractions that contained as little as 4 × 10−10m of the protein complex, indicating a dissociation constant of the complex that is considerably lower than this protein concentration. When the Rad7-Rad16 protein complex from the last step of purification in Mono S (Fraction VI) was subjected to SDS-polyacrylamide gel electrophoresis and staining with Coomassie Blue, only the Rad7 and Rad16 bands were seen (Fig. 1, B andC), indicating a high degree of purity of the complex. Image analysis of the gel in Fig. 1 B revealed a one to one stoichiometry of the Rad7 and Rad16 proteins in the complex. We speculated that NEF4 might effect the repair of transcriptionally inactive DNA by sensing the DNA damage located in such regions and then recruiting the basic NER machinery to initiate the repair reaction. As a first test of this hypothesis, we examined whether NEF4 would bind UV-damaged DNA using a DNA mobility shift assay. The DNA fragment was labeled with 32P, irradiated with UV doses ranging from 1 to 12 kJ/m2, and then incubated with NEF4 in the presence of ATP and an excess of unlabeled φX174 DNA at 30 °C. After running the reaction mixtures in polyacrylamide gels under nondenaturing conditions, the gels were dried and exposed to x-ray films to reveal the radiolabeled DNA species. As shown in Fig. 2, incubation of the UV-irradiated DNA probe with NEF4 resulted in the formation of slower migrating forms of DNA, indicating binding of the damaged DNA by NEF4; no binding of NEF4 to the undamaged DNA was seen. The level of nucleoprotein complex formation was proportional to the amount of NEF4 (Fig. 2, Aand B) and to the UV dose (Fig. 2, C andD). Multiple nucleoprotein complexes were detected in these experiments, with the slower migrating nucleoprotein species being more prevalent at higher UV doses and with increasing concentrations of NEF4 (Fig. 2), suggesting that these species contained multiple NEF4 molecules bound to different damage sites in the DNA probe. Importantly, when ATP was omitted from the reaction, binding of the UV-damaged DNA decreased dramatically, such that only ∼6% of the UV-irradiated DNA was bound by NEF4 in the absence of the nucleotide, as compared with greater than 50% binding in its presence (Fig. 2 E). Although NEF4 possesses an ATPase activity that is activated by either single-stranded or double-stranded DNA, 2S. N. Guzder, unpublished observations. ATP binding by NEF4 is apparently sufficient for damage recognition, because the nonhydrolyzable ATP analogue ATPγS is nearly as effective as ATP in promoting damage binding (Fig. 2 E). Having established that NEF4 has high affinity for UV-damaged DNA, we then tested if NEF4 might improve the efficiency of the damage-specific incision reaction mediated by the basic NER machinery comprising NEF1, NEF2, NEF3, and RPA (2Guzder S.N. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1996; 271: 8903-8910Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 3Guzder S.N. Habraken Y. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1995; 270: 12973-12976Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 4Habraken Y. Sung P. Prakash S. Prakash L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10718-10722Crossref PubMed Scopus (45) Google Scholar, 15Sung P. Guzder S.N. Prakash L. Prakash S. J. Biol. Chem. 1996; 271: 10821-10826Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). To do this, we examined the rate of incision of supercoiled M13 DNA that had been exposed to 30 J/m2 UV light to introduce on average 1.8 photoproducts per plasmid DNA molecule (approximately 7.3 kilobase pairs long) by the basic NER machinery with or without added purified NEF4. Interestingly, addition of NEF4 to the in vitro NER reaction resulted in a marked stimulation of the conversion of the UV-irradiated supercoiled M13 plasmid DNA to the open circular form (Fig. 3, A and B), while no incision of the nondamaged M13 DNA was observed whether or not NEF4 was added to the repair reaction (Fig. 3 A). These observations indicate that NEF4 helps promote the damage-specific incision reaction. We also subjected the NER reaction to labeling with [α-32P]ddATP and calf thymus terminal transferase to detect the excision DNA fragments (3Guzder S.N. Habraken Y. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1995; 270: 12973-12976Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 15Sung P. Guzder S.N. Prakash L. Prakash S. J. Biol. Chem. 1996; 271: 10821-10826Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). In agreement with the results from the agarose gel method, the amount of excision fragments generated was also higher when NEF4 was included in the repair reaction (data not shown). Our findings with NEF4 suggest that eukaryotes employ different mechanisms of damage recognition for the nontranscribedversus the transcribed strand. NEF4, an ATP-dependent damage recognition factor, is essential for the repair of the nontranscribed strand, whereas the products of theCSA and CSB/RAD26 genes are essential for preferential repair of the transcribed strand. NEF4 may be pivotal in locating the damage on the nontranscribed strand and in transcriptionally inactive regions of the genome, and it may do so by a tracking mechanism that utilizes the energy of ATP hydrolysis. Subsequent to binding the DNA lesion, NEF4 may serve as the nucleation site for the assembly of the other repair components for dual incision to occur. An additional role of NEF4 in the turnover of the incision enzyme complex is also possible. We thank Yvette Habraken for help in preparation of the manuscript and E. J. Miller and T. Johnson for technical assistance." @default.
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W2041261463 title "Yeast Rad7-Rad16 Complex, Specific for the Nucleotide Excision Repair of the Nontranscribed DNA Strand, Is an ATP-dependent DNA Damage Sensor" @default.
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