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• W2040779655 abstract "Nucleotide excision repair (NER) is the primary mechanism by which both Saccharomyces cerevisiae and human cells remove the DNA lesions caused by ultraviolet light and other mutagens. This complex process involves the coordinated actions of more than 20 polypeptides. To facilitate biochemical studies of NER in yeast, we have established a simple protocol for preparing whole cell extracts which perform NER in vitro. As expected, this assay of in vitro repair was dependent on the products of RAD genes such as RAD14, RAD4, and RAD2. Interestingly, it was also dependent upon proteins encoded by the RAD7, RAD16, and RAD23 genes whose precise roles in NER are uncertain, but not the RAD26 gene whose product is believed to participate in coupling NER to transcription. Replication protein A (RPA/Rpa), known to be required for NER in human cell extracts, was also shown by antibody inhibition and immunodepletion experiments to be required for NER in our yeast cell extracts. Moreover, yeast cells with temperature-sensitive mutations in the RFA2 gene, which encodes the 34-kDa subunit of Rpa, had increased sensitivity to UV and yielded extracts defective in NER in vitro. These data indicate that Rpa is an essential component of the NER machinery in S. cerevisiae as it is in mammalian cells. Nucleotide excision repair (NER) is the primary mechanism by which both Saccharomyces cerevisiae and human cells remove the DNA lesions caused by ultraviolet light and other mutagens. This complex process involves the coordinated actions of more than 20 polypeptides. To facilitate biochemical studies of NER in yeast, we have established a simple protocol for preparing whole cell extracts which perform NER in vitro. As expected, this assay of in vitro repair was dependent on the products of RAD genes such as RAD14, RAD4, and RAD2. Interestingly, it was also dependent upon proteins encoded by the RAD7, RAD16, and RAD23 genes whose precise roles in NER are uncertain, but not the RAD26 gene whose product is believed to participate in coupling NER to transcription. Replication protein A (RPA/Rpa), known to be required for NER in human cell extracts, was also shown by antibody inhibition and immunodepletion experiments to be required for NER in our yeast cell extracts. Moreover, yeast cells with temperature-sensitive mutations in the RFA2 gene, which encodes the 34-kDa subunit of Rpa, had increased sensitivity to UV and yielded extracts defective in NER in vitro. These data indicate that Rpa is an essential component of the NER machinery in S. cerevisiae as it is in mammalian cells. Nucleotide excision repair (NER) 1The abbreviations used are: NERnucleotide excision repairRpareplication protein A (yeast)RPAreplication protein A (human)AAAFN-acetoxy-2-acetylaminofluorenePAGEpolyacrylamide gel electrophoresisXPxeroderma pigmentosumUVultraviolet light. is a versatile DNA repair strategy found ubiquitously in prokaryotes and eukaryotes. NER is capable of removing a broad spectrum of DNA lesions caused by physical and chemical mutagens (1Prakash S. Sung P. Prakash L. Annu. Rev. Genet. 1993; 27: 33-70Crossref PubMed Scopus (257) Google Scholar, 2Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. ASM Press, Washington, D. C.1995Google Scholar, 3Sancar A. J. Biol. Chem. 1995; 270: 15915-15918Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 4Aboussekhra A. Wood R.D. Curr. Opin. Genet. Dev. 1994; 4: 212-220Crossref PubMed Scopus (74) Google Scholar, 5Lehmann A.R. Trends Biochem. Sci. 1995; 20 (1995): 402-405Abstract Full Text PDF PubMed Scopus (137) Google Scholar). Failure to remove DNA lesions from the genome as a result of defective NER may lead to cancer-susceptibility, as exemplified by the hereditary human disease xeroderma pigmentosum (XP) (2Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. ASM Press, Washington, D. C.1995Google Scholar, 3Sancar A. J. Biol. Chem. 1995; 270: 15915-15918Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 4Aboussekhra A. Wood R.D. Curr. Opin. Genet. Dev. 1994; 4: 212-220Crossref PubMed Scopus (74) Google Scholar, 5Lehmann A.R. Trends Biochem. Sci. 1995; 20 (1995): 402-405Abstract Full Text PDF PubMed Scopus (137) Google Scholar). NER involves the concerted actions of several different enzymatic activities and can be arbitrarily divided into the following steps: damage recognition, incision and excision of the lesion and its flanking DNA, and repair DNA synthesis to fill in the resulting single-stranded gap. Many of the proteins encoded by XP genes and their evolutionarily conserved RAD gene homologs in Saccharomyces cerevisiae are now known to function in the early steps of excision repair, participating in the removal of DNA lesions prior to the repair DNA synthesis step. Some of the proteins involved in the repair DNA synthesis step of excision repair also function in the replication of cellular DNA and include proteins such as proliferating cell nuclear antigen (6Shivji M.K.K. Kenny M.K. Wood R.D. Cell. 1992; 69: 367-374Abstract Full Text PDF PubMed Scopus (734) Google Scholar, 7Nichols A.F. Sancar A. Nucleic Acids Res. 1992; 20: 3559-3564Crossref Scopus (3) Google Scholar, 8Ayyagari R. Impellizzeri K.J. Yoder B.L. Gary S.L. Burgers P.M.J. Mol. Cell. Biol. 1995; 15: 4420-4429Crossref PubMed Scopus (187) Google Scholar) and replication protein A (9Coverley D. Kenny M.K. Munn M. Rupp W.D. Lane D.P. Wood R.D. Nature. 1991; 349: 538-541Crossref PubMed Scopus (198) Google Scholar, 10Coverley D. Kenny M.K. Lane D.P. Wood R.D. Nucleic Acids Res. 1992; 20: 3873-3880Crossref PubMed Scopus (137) Google Scholar, 11Aboussekhra A. Biggerstaff M. Shivji M.K.K. Vilpo J.A. Moncollin V. Podust V.N. Protic M. Hubscher U. Egly J.-M. Wood R.D. Cell. 1995; 80: 859-868Abstract Full Text PDF PubMed Scopus (751) Google Scholar, 12Mu 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 (409) Google Scholar, 13Guzder 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, 14Shivji M.K.K. Podust V.N. Hubscher U. Wood R.D. Biochemistry. 1995; 34: 5011-5017Crossref PubMed Scopus (241) Google Scholar). nucleotide excision repair replication protein A (yeast) replication protein A (human) N-acetoxy-2-acetylaminofluorene polyacrylamide gel electrophoresis xeroderma pigmentosum ultraviolet light. Both yeast and mammalian replication protein A (Rpa/RPA) are trimeric complexes consisting of polypeptides of approximately 70, 34, and 14 kDa (15Wold M.S. Kelly T.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2523-2527Crossref PubMed Scopus (369) Google Scholar, 16Fairman M.P. Stillman B. EMBO J. 1988; 7: 1211-1218Crossref PubMed Scopus (295) Google Scholar, 17Heyer W.-D. Kolodner R.D. Biochemistry. 1989; 28: 2856-2862Crossref PubMed Scopus (33) Google Scholar, 18Brill S.J. Stillman B. Nature. 1989; 342: 92-95Crossref PubMed Scopus (189) Google Scholar, 19Brill S.J. Stillman B. Genes Dev. 1991; 5: 1589-1600Crossref PubMed Scopus (191) Google Scholar). RPA, a single-stranded DNA-binding protein, might function in the DNA synthesis step of NER as it does in cellular and viral DNA replication. Recent studies (11Aboussekhra A. Biggerstaff M. Shivji M.K.K. Vilpo J.A. Moncollin V. Podust V.N. Protic M. Hubscher U. Egly J.-M. Wood R.D. Cell. 1995; 80: 859-868Abstract Full Text PDF PubMed Scopus (751) Google Scholar, 12Mu 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 (409) Google Scholar, 13Guzder 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, 20He Z. Henricksen L.A. Wold M.S. Ingles C.J. Nature. 1995; 374: 566-568Crossref PubMed Scopus (374) Google Scholar, 21Li L. Lu X. Peterson C.A. Legerski R.J. Mol. Cell. Biol. 1995; 15: 5396-5402Crossref PubMed Scopus (228) Google Scholar, 22Matsuda T. Saijo M. Kuraoka I. Kobayashi T. Nakatsu Y. Nagai A. Enjoji T. Masutani C. Sugasawa K. Hanaoka F. Yasui A. Tanaka K. J. Biol. Chem. 1995; 270: 4152-4157Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar) have suggested, however, that RPA participates in both the early (damage recognition, incision, excision) steps as well as the late (repair synthesis) step of NER. The human RPA complex is now known to interact directly with XPA (20He Z. Henricksen L.A. Wold M.S. Ingles C.J. Nature. 1995; 374: 566-568Crossref PubMed Scopus (374) Google Scholar, 21Li L. Lu X. Peterson C.A. Legerski R.J. Mol. Cell. Biol. 1995; 15: 5396-5402Crossref PubMed Scopus (228) Google Scholar, 22Matsuda T. Saijo M. Kuraoka I. Kobayashi T. Nakatsu Y. Nagai A. Enjoji T. Masutani C. Sugasawa K. Hanaoka F. Yasui A. Tanaka K. J. Biol. Chem. 1995; 270: 4152-4157Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 23Lee S.-H. Kim D.-K. Drissi R. J. Biol. Chem. 1995; 270: 21800-21805Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), the human homolog of the yeast damage-recognition protein Rad14 (24Bankmann M. Prakash L. Prakash S. Nature. 1992; 355: 555-558Crossref PubMed Scopus (97) Google Scholar, 25Tanaka K. Miura N. Satokata I. Miyamoto I. Yoshida M.C. Satoh S. Kondo A. Yasui A. Okayama H. Okada Y. Nature. 1990; 348: 73-76Crossref PubMed Scopus (344) Google Scholar). This interaction enhances the ability of the RPA-XPA complex to bind to damaged DNA (20He Z. Henricksen L.A. Wold M.S. Ingles C.J. Nature. 1995; 374: 566-568Crossref PubMed Scopus (374) Google Scholar, 21Li L. Lu X. Peterson C.A. Legerski R.J. Mol. Cell. Biol. 1995; 15: 5396-5402Crossref PubMed Scopus (228) Google Scholar). Following the damage-recognition step, incisions flanking the lesion are made by two endonuclease activities, in humans XPG (26Scherly D. Nouspikel T. Corlet J. Ucla C. Bairoch A. Clarkson S.G. Nature. 1993; 363: 182-185Crossref PubMed Scopus (181) Google Scholar, 27O'Donovan A Davies A.A. Moggs J.G. West S.C. Wood R.D. Nature. 1993; 371: 432-435Crossref Scopus (398) Google Scholar) and the ERCC1-XPF complex (28Biggerstaff M. Szymkowski D.E. Wood R.D. EMBO J. 1993; 12: 3685-3692Crossref PubMed Scopus (149) Google Scholar, 29Van Vuuren A.J. Appeldoorn E. Odijk H. Yasui A. Jaspers N.G.J. Hoeijmakers J.H.J. EMBO J. 1993; 12: 3693-3701Crossref PubMed Scopus (142) Google Scholar, 30Park C.-H. Bessho T. Matsunaga T. Sancar A. J. Biol. Chem. 1995; 270: 22657-22660Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), and in yeast Rad2 (31Habraken Y. Sung P. Prakash L. Prakash S. Nature. 1993; 366: 365-368Crossref PubMed Scopus (115) Google Scholar) and Rad1-Rad10 (32Tomkinson A.E. Bardwell A.J. Bardwell L. Tappe N.J. Friedberg E.C. Nature. 1993; 362: 860-862Crossref PubMed Scopus (158) Google Scholar, 33Sung P. Reynolds P. Prakash L. Prakash S. J. Biol. Chem. 1993; 268: 26391-26399Abstract Full Text PDF PubMed Google Scholar). Two helicase activities encoded by the XPD and XPB genes (34Sung P. Bailly V. Weber C. Thompson L.H. Prakash L. Prakash S. Nature. 1993; 365: 852-855Crossref PubMed Scopus (286) Google Scholar, 35Schaeffer L. Roy R. Humbert S. Moncollin V. Vermeulen W. Hoeijmakers J.H.J. Chambon P. Egly J.M. Science. 1993; 260: 58-63Crossref PubMed Scopus (666) Google Scholar) in humans and RAD3 and RAD25 in S. cerevisiae (36Sung P. Prakash L. Matson S.W. Prakash S. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8951-8955Crossref PubMed Scopus (167) Google Scholar, 37Harosh I. Naumovski L. Friedberg E.C. J. Biol. Chem. 1989; 264: 20532-20539Abstract Full Text PDF PubMed Google Scholar, 38Gulyas K.D. Donahue T.F. Cell. 1992; 69: 1031-1042Abstract Full Text PDF PubMed Scopus (109) Google Scholar, 39Park E. Guzder S.N. Koken M.H.M. Jaspers-Dekker I. Weeda G. Hoeijmakers J.H.J. Prakash L. Prakash S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11416-11420Crossref PubMed Scopus (112) Google Scholar) are also involved in the incision/excision steps prior to repair synthesis. In addition to proliferating cell nuclear antigen and RPA, repair DNA synthesis also involves the participation of replication factor C (11Aboussekhra A. Biggerstaff M. Shivji M.K.K. Vilpo J.A. Moncollin V. Podust V.N. Protic M. Hubscher U. Egly J.-M. Wood R.D. Cell. 1995; 80: 859-868Abstract Full Text PDF PubMed Scopus (751) Google Scholar, 14Shivji M.K.K. Podust V.N. Hubscher U. Wood R.D. Biochemistry. 1995; 34: 5011-5017Crossref PubMed Scopus (241) Google Scholar), polymerase δ or ϵ (11Aboussekhra A. Biggerstaff M. Shivji M.K.K. Vilpo J.A. Moncollin V. Podust V.N. Protic M. Hubscher U. Egly J.-M. Wood R.D. Cell. 1995; 80: 859-868Abstract Full Text PDF PubMed Scopus (751) Google Scholar, 14Shivji M.K.K. Podust V.N. Hubscher U. Wood R.D. Biochemistry. 1995; 34: 5011-5017Crossref PubMed Scopus (241) Google Scholar, 40Budd M.E. Campbell J.L. Mol. Cell. Biol. 1995; 15: 2173-2179Crossref PubMed Scopus (90) Google Scholar), and one of the DNA ligases (11Aboussekhra A. Biggerstaff M. Shivji M.K.K. Vilpo J.A. Moncollin V. Podust V.N. Protic M. Hubscher U. Egly J.-M. Wood R.D. Cell. 1995; 80: 859-868Abstract Full Text PDF PubMed Scopus (751) Google Scholar, 14Shivji M.K.K. Podust V.N. Hubscher U. Wood R.D. Biochemistry. 1995; 34: 5011-5017Crossref PubMed Scopus (241) Google Scholar). Biochemical analyses of NER have depended largely on the availability of cell-free extracts that can support excision repair. The in vitro system developed by Wood and his colleagues (41Wood R.D. Robins P. Lindahl T. Cell. 1988; 53: 97-106Abstract Full Text PDF PubMed Scopus (380) Google Scholar) has proven to be instrumental in the dissection of the NER pathway in human cells. On the other hand, understanding excision repair in S. cerevisiae has relied mainly on the genetic analyses of rad mutants. The potential for biochemical analysis of NER in yeast cells has not yet been fully realized owing in part to the lack of a simple in vitro repair system. The only existing cell-free system from S. cerevisiae, that described by Wang et al. (42Wang Z. Wu X. Friedberg E.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4907-4911Crossref PubMed Scopus (73) Google Scholar, 43Wang Z. Buratowski S. Svejstrup J.Q. Feaver W.J. Wu X. Kornberg R.D. Donahue T.F. Friedberg E.C. Mol. Cell. Biol. 1995; 15: 2288-2293Crossref PubMed Scopus (75) Google Scholar), involves the preparation of separate nuclear and whole cell extracts. Hence, it seemed desirable to develop a simple in vitro preparation from yeast cells capable of NER and applicable for use with a variety of different yeast strains. Here we report the development of such a system using a whole cell extract prepared from S. cerevisiae. We have utilized this in vitro system to demonstrate a requirement for the products of the genes RAD7, RAD16, and RAD23. Furthermore, we have also exploited this in vitro repair system to assess the role of yeast Rpa in NER. In addition, yeast strains with point mutations in the RFA2 gene were constructed and characterized. Taken together, our biochemical and genetic results indicate that, as in human cells, Rpa plays an important role in the process of NER in S. cerevisiae cells. The S. cerevisiae strains used in this study were BJ2168 (from Dr. J. Segall, University of Toronto), LP2899, a gift of Dr. L. Prakash, and rad mutant strains, MGSC131 (rad4Δ::URA3), MGSC139 (rad14Δ::LEU2), MGSC104 (rad7Δ::LEU2), W303236 (rad16Δ::URA3), MGSC101 (rad23Δ::URA3), MGSC102 (rad26Δ::HIS3) and their isogenic parental strain W303-1B (44Verhage R.A. van Gool A.J. de Groot N. Hoeijmakers J.H.J. van de Putte P. Brouwer J. Mol Cell. Biol. 1996; 16: 496-502Crossref PubMed Scopus (91) Google Scholar) (kindly provided by Dr. J. Brouwer, Leiden University), and EMY75 (rad2Δ::URA3 from L. Prakash). The procedure for the preparation of NER-proficient yeast extract was a modification of protocols originally intended for transcription studies (45Schultz M.C. Choe S.Y. Reeder R.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1004-1008Crossref PubMed Scopus (83) Google Scholar). Yeast cultures were grown at 27°C in complete medium (YEPD: 1% yeast extract, 2% Bacto-Peptone, 2% glucose) with vigorous shaking. Cells were harvested at an OD600 of 2 by first chilling in ice water and then centrifuging at 4000 rpm for 4 min in a Sorval H-6000A rotor. The cells were then washed once in ice-cold water and once in extraction buffer (0.2 M Tris, pH 7.5, 0.39 M (NH4)2SO4, 10 mM MgSO4, 20% (v/v) glycerol, 1 mM EDTA, 1 mM dithiothreitol) containing various protease inhibitors (phenymethylsulfonyl fluoride, 1 mM; benzamidine hydrochloride, 2 mM; pepstatin A, 3.5 μg/ml; leupeptin, 1 μg/ml; bestatin, 0.35 μg/ml; and aprotinin, 10 μg/ml). The drained cell pellet was scraped into a syringe, then extruded directly into liquid nitrogen and stored at −70°C. Frozen cells were broken by manual grinding under liquid nitrogen using a ceramic mortar and pestle. Grinding continued until the material was reduced to powder. After grinding, the frozen powder of broken cells was mixed with 1 volume of cold extraction buffer supplemented with protease inhibitors and allowed to thaw at 4°C. The cell lysate was then centrifuged at 120,000 × g for 2 h at 4°C. The clear supernatant was recovered and (NH4)2SO4 added to 2.94 M by the addition of 337 mg of solid (NH4)2SO4/ml of lysate over the course of 1 h. The suspension was stirred for another 30 min, and the precipitated protein pelleted by centrifugation at 40,000 × g for 15 min. The pellet was resuspended in a small volume (approximately 50 μl/g of cells) of dialysis buffer (20 mM HEPES pH 7.5, 20% (v/v) glycerol, 10 mM MgSO4, 10 mM EGTA, 5 mM dithiothreitol) plus protease inhibitors. The sample was then dialyzed against the same buffer plus 1 mM phenymethylsulfonyl fluoride for 12-16 h. The dialysate was centrifuged to remove precipitated protein and the resulting supernatant was collected and stored at −70°C until use. Protein concentrations were determined by the Bio-Rad colorimetric assay using bovine serum albumin as standard. DNA from plasmids pUC18 (2.7 kilobase pairs) and pGEM-3Zf(+) (3.2 kilobase pairs) was isolated by alkaline lysis and CsCl-ethidium bromide equilibrium centrifugation. The pUC18 DNA was treated with N-acetoxy-2-acetylaminofluorene (AAAF) and repurified on a 5-20% sucrose gradient as described by Wang et al. (42Wang Z. Wu X. Friedberg E.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4907-4911Crossref PubMed Scopus (73) Google Scholar). Reaction mixtures (50 μl) contained 300 ng of AAAF-treated pUC18 and 300 ng of control pGEM-3Zf(+) DNA, 45 mM HEPES-KOH (pH 7.8), 70 mM KCl, 7.4 mM MgCl2, 0.9 mM dithiothreitol, 0.4 mM EDTA, 20 μM each of dGTP, dATP, TTP, 8 μM dCTP, 2 μCi of [α-32P]dCTP (3000 Ci/mmol), 2 mM ATP, 40 mM disodium phosphocreatine, 2.5 μg of creatine kinase, 3.4% glycerol, 18 μg of bovine serum albumin, and 250 μg of protein as yeast whole cell extract (typically 6-8 μl). Reactions were incubated at 28°C for 2 h. Plasmid DNA was purified from the reaction mixtures, linearized by digestion with HindIII, analyzed on a 1% agarose gel, and autoradiographed as described by Wang et al. (42Wang Z. Wu X. Friedberg E.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4907-4911Crossref PubMed Scopus (73) Google Scholar). To quantitate the extent of repair synthesis, dried gels were exposed to either storage Phosphor screens or autoradiography films. The amount of radioactivity in each band was quantitated by phosphoimaging analysis or densitometry and comparison to known radioactivity standards. For the antibody inhibition experiments shown in Fig. 4, the indicated amount of preimmune or anti-yeast Rpa antiserum was preincubated with the cell extracts for 15 min at 28°C before addition of reaction buffer and plasmid DNAs. The polyclonal antibodies against yeast Rpa used in these studies were raised in a rabbit by injection of recombinant yeast Rpa protein. The depletion of yeast Rpa from whole cell extracts was effected using an immunodepletion procedure described by Adachi and Laemmli (46Adachi Y. Laemmli U.K. EMBO J. 1994; 13: 4153-4164Crossref PubMed Scopus (110) Google Scholar). Briefly, phosphate-buffered saline-washed Protein A-Sepharose CL-4B matrix was incubated with equal amounts of preimmune or anti-yeast Rpa serum at room temperature for 2 h. The matrices were washed and incubated with 4 volumes of yeast cell extract in the presence of an ATP-regenerating system (46Adachi Y. Laemmli U.K. EMBO J. 1994; 13: 4153-4164Crossref PubMed Scopus (110) Google Scholar) at 4°C for 1 h. The Sepharose was then briefly pelleted in an Eppendorf tube and the supernatant was collected as the depleted extract. As estimated by a Western blot, more than 90% of the yeast Rpa protein in the extract was removed (data not shown). Plasmids pJM124, pJM223, and pJM329 contain, respectively, the RFA1 gene, RFA2 cDNA, and the RFA3 gene under the control of the phage T7 promoter of plasmid pET11a (47Studier F. Rosenberg H. Dunn J. Dubendorff J. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (6006) Google Scholar) and are described in Ref. 19Brill S.J. Stillman B. Genes Dev. 1991; 5: 1589-1600Crossref PubMed Scopus (191) Google Scholar. The T7 promoter, gene, and transcription terminator of these plasmids can be moved as a BamHI/BglII cassette. The 1.2-kilobase pair BamHI/BglII cassette of pJM223 was inserted into the BamHI site of pJM329 to create pJM332. The resulting 1.9-kilobase pair BamHI/BglII cassette of pJM332 was then inserted into the BglII site of pJM124 to create pJM126 containing the three RFA genes on a single plasmid, each with its own T7 promoter. pJM126 was transformed into BL21(DE3) for protein expression. BL21(DE3) cells containing pJM126 were grown in LB containing 0.1 mg/ml ampicillin at 22-37°C to an OD of 0.5-0.8. Isopropyl-1-thio-β-D-galactopyranoside was then added to 0.4 mM and the induction continued for 3 h. Cells were harvested and the bacterial pellet resuspended in one-tenth volume of A buffer (18Brill S.J. Stillman B. Nature. 1989; 342: 92-95Crossref PubMed Scopus (189) Google Scholar) with 50 mM NaCl and 1 mg/ml lysozyme. Following incubation at 4°C for 15 min, Nonidet P-40 was added to a final concentration of 0.1%. The lysate was subjected to a total of 3 freeze-thaw cycles using dry ice/ethanol and swirling in a 37°C bath. Chromosomal DNA was dispersed by sonication (3 × 1 min) in volumes of approximately 20 ml on ice. The extract was clarified by centrifugation at 20,000 × g for 20 min at 4°C. The extract produced from 1 liter of culture was loaded on a 100-ml phosphocellulose column equilibrated in buffer A with 50 mM NaCl and the column washed with 300 ml of buffer A with 50 mM NaCl. Escherichia coli SSB is found in the flow-through of this column. Rpa was eluted with a 600-ml gradient from 50 to 800 mM NaCl in buffer A and assayed for unwinding activity as described previously (48Tsurimoto T. Fairman M.P. Stillman B. Mol. Cell. Biol. 1989; 9: 3839-3849Crossref PubMed Scopus (62) Google Scholar). Rpa elutes at approximately 200 mM NaCl from this column. Active fractions were pooled, NaCl added to 500 mM, and the sample loaded onto a 5-ml ssDNA-cellulose column equilibrated in buffer A with 500 mM NaCl. This column was washed with 15 ml of buffer A with 500 mM NaCl, 25 ml of buffer A with 750 mM NaCl, and 25 ml of buffer A with 1.5 M NaCl and 50% ethylene glycol. The protein peak from this last step contains approximately 1 mg of recombinant Rpa from a 1-liter culture. Approximately 50 μg of recombinant Rpa was then loaded onto a 5-ml 15-35% glycerol gradient in buffer A containing 100 mM NaCl. A parallel gradient contained a similar amount of Rpa purified from yeast. The gradients were centrifuged in an SW50.1 rotor at 49,000 rpm for 21 h. Fractions (130 μl) were collected from both gradients, 8-μl aliquots assayed for unwinding activity and 14-μl aliquots subjected to SDS-PAGE followed by silver staining. The unwinding assay was performed essentially as described by Tsurimoto et al. (48Tsurimoto T. Fairman M.P. Stillman B. Mol. Cell. Biol. 1989; 9: 3839-3849Crossref PubMed Scopus (62) Google Scholar). A final reaction volume of 20 μl containing 30 mM HEPES-KOH (pH 8.0), 7 mM MgCl2, 0.5 mM dithiothreitol, 4 mM ATP, 40 mM creatine phosphate, 0.4 μg of creatine phosphokinase, 0.4 μg of bovine serum albumin, 0.08 μg of calf thymus topoisomerase I, 0.3 μg of pSV011 plasmid DNA, 0.5 μg of SV40 T antigen, and the indicated amounts of Rpa was assembled on ice and then placed at 37°C for 15 min. The reaction was stopped by placing it on ice. Four μl of a solution of 4% SDS, 40 mM EDTA, and 6 mg/ml Pronase was added and the tubes placed at 37°C for 30 min. The reaction products were then directly resolved by electrophoresis in a 0.8% agarose gel, stained with ethidium bromide, and photographed. For experiments employing recombinant Rpa in the in vitro assays of nucleotide excision repair, recombinant Rpa protein was purified through an Affi-Gel Blue column, an hydroxylapatite column, and an ssDNA-cellulose column as described for the purification of human RPA by Henricksen et al. (49Henricksen L.A. Umbricht C.B. Wold M.S. J. Biol. Chem. 1994; 269: 11121-11132Abstract Full Text PDF PubMed Google Scholar). Details concerning the identification and characterization of rfa2 mutants will be presented elsewhere. 2H. S. Maniar and S. J. Brill, manuscript in preparation. Briefly, the entire RFA2 gene on plasmid pJM215 (19Brill S.J. Stillman B. Genes Dev. 1991; 5: 1589-1600Crossref PubMed Scopus (191) Google Scholar) was subjected to 35 cycles of amplification with Taq DNA polymerase in the presence of 1 mM MnCl2 and the universal forward and reverse sequencing primers. The polymerase chain reaction product was digested with BamHI and SalI and ligated into the yeast centromeric plasmid pRS415. This library of mutagenized RFA2 plasmid DNA was amplified in bacteria and introduced into strain SBY105 which carries a deletion of the RFA2 gene but is kept alive by plasmid pJM218 (19Brill S.J. Stillman B. Genes Dev. 1991; 5: 1589-1600Crossref PubMed Scopus (191) Google Scholar) carrying the RFA2 cloned into the yeast centromeric plasmid YCp50. Transformants were replica-plated to plates containing the drug 5-fluoroorotic acid and placed at 37°C and 25°C. Strains showing no growth after 2 days at 37°C were identified and the RFA2 plasmid rescued from the corresponding 25°C colony by transformation into E. coli. Mutant plasmid DNA was then reintroduced into strain SBY105 (19Brill S.J. Stillman B. Genes Dev. 1991; 5: 1589-1600Crossref PubMed Scopus (191) Google Scholar) to confirm that the temperature-sensitive growth defect was plasmid-dependent. Strains passing this test were tested for loss of viability in a liquid culture at 37°C. Typically these ts strains showed 10-20% viability following 4 h exposure to 37°C. Cells were grown at 25°C overnight in YPD medium, diluted in sterile water, and plated in duplicate on YPD plates and immediately irradiated with a Stratalinker UV cross-linker (Stratagene). The plates were then incubated at 25°C for 3 days and surviving colonies were counted. The availability of human cell-free extracts capable of performing nucleotide excision repair (41Wood R.D. Robins P. Lindahl T. Cell. 1988; 53: 97-106Abstract Full Text PDF PubMed Scopus (380) Google Scholar) has permitted major advances in our understanding of this pathway of DNA repair. The genetic analysis of the sensitivity to UV of S. cerevisiae has also provided an excellent approach for studying NER in eukaryotes (1Prakash S. Sung P. Prakash L. Annu. Rev. Genet. 1993; 27: 33-70Crossref PubMed Scopus (257) Google Scholar, 2Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. ASM Press, Washington, D. C.1995Google Scholar). However, the full potential of using S. cerevisiae and its many mutant rad strains in studies of NER has not been fully exploited. Biochemical analyses of NER in yeast may have been hampered by the lack of a simple method of preparing yeast extracts capable of repair in vitro. As the human extract system was developed originally for studying in vitro transcription, we tested whether yeast extracts capable of in vitro transcription (45Schultz M.C. Choe S.Y. Reeder R.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1004-1008Crossref PubMed Scopus (83) Google Scholar) would also perform nucleotide excision repair. In this protocol, yeast cells were quickly frozen and then ground in liquid nitrogen. After addition of minimal amounts of buffer, the thawed extract was then clarified by a centrifugation step, further concentrated by ammonium sulfate precipitation, solubilized, and dialyzed (see “Experimental Procedures”). Nucleotide excision repair in the yeast cell extracts was monitored by the incorporation of radiolabeled nucleotides into plasmid DNA during repair DNA synthesis. DNA damage was introduced into plasmid substrates by treatment with AAAF which forms DNA adducts known to be corrected by the excision repair pathway (50Hansson J. Munn M. Rupp W.D. Kahn R. Wood R.D. J. Biol. Chem. 1989; 264: 21788-21792Abstract Full Text PDF PubMed Google Scholar). An untreated plasmid was also included in each reaction to monitor repair-independent nucleotide incorporation. As shown in Fig. 1A, damage-dependent repair synthesis was detected in extracts made from three commonly used laboratory S. cerevisiae strains. The extent of damage-dependent repair synthesis increased with the amount of protein added. An amount of extract corresponding to 250 μg of protein in an assay volume of 50 μl produced good signals above the background level of incorporation of radiolabeled nucleotide into the unt" @default.
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• W2040779655 title "Assessing the Requirements for Nucleotide Excision Repair Proteins of Saccharomyces cerevisiae in an in Vitro System" @default.
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