The Pso4 mRNA Splicing and DNA Repair Complex Interacts with WRN for Processing of DNA Interstrand Cross-links
2005; Elsevier BV; Volume: 280; Issue: 49 Linguagem: Inglês
10.1074/jbc.m508453200
ISSN1083-351X
AutoresNianxiang Zhang, Ramandeep Kaur, Xiaoyan Lu, Xi Shen, Lei Li, Randy J. Legerski,
Tópico(s)Genomics and Chromatin Dynamics
ResumoDNA interstrand cross-links (ICLs) are perhaps the most formidable lesion encountered by the cellular DNA repair machinery, and the elucidation of the process by which they are removed in eukaryotic cells has proved a daunting task. In particular, the early stages of adduct recognition and uncoupling of the cross-link have remained elusive principally because genetic studies have not been highly revealing. We have developed a biochemical assay in which processing of a DNA substrate containing a site-specific psoralen ICL can be monitored in vitro. Using this assay we have shown previously that the mismatch repair factor MutSβ, the nucleotide excision repair heterodimer Ercc1-Xpf, and the replication proteins RPA and PCNA are involved in an early stage of psoralen ICL processing. Here, we report the identification of two additional factors required in the ICL repair process, a previously characterized pre-mRNA splicing complex composed of Pso4/Prp19, Cdc5L, Plrg1, and Spf27 (Pso4 complex), and WRN the protein deficient in Werner syndrome. Analysis of the WRN protein indicates that its DNA helicase function, but not its exonuclease activity, is required for ICL processing in vitro. In addition, we show that WRN and the Pso4 complex interact through a direct physical association between WRN and Cdc5L. A putative model for uncoupling of ICLs in mammalian cells is presented. DNA interstrand cross-links (ICLs) are perhaps the most formidable lesion encountered by the cellular DNA repair machinery, and the elucidation of the process by which they are removed in eukaryotic cells has proved a daunting task. In particular, the early stages of adduct recognition and uncoupling of the cross-link have remained elusive principally because genetic studies have not been highly revealing. We have developed a biochemical assay in which processing of a DNA substrate containing a site-specific psoralen ICL can be monitored in vitro. Using this assay we have shown previously that the mismatch repair factor MutSβ, the nucleotide excision repair heterodimer Ercc1-Xpf, and the replication proteins RPA and PCNA are involved in an early stage of psoralen ICL processing. Here, we report the identification of two additional factors required in the ICL repair process, a previously characterized pre-mRNA splicing complex composed of Pso4/Prp19, Cdc5L, Plrg1, and Spf27 (Pso4 complex), and WRN the protein deficient in Werner syndrome. Analysis of the WRN protein indicates that its DNA helicase function, but not its exonuclease activity, is required for ICL processing in vitro. In addition, we show that WRN and the Pso4 complex interact through a direct physical association between WRN and Cdc5L. A putative model for uncoupling of ICLs in mammalian cells is presented. DNA interstrand cross-linking agents such as cyclophosamide and mitomycin C are widely used as chemotherapeutic agents in the treatment of a broad spectrum of cancers. The clinical usage of these drugs has fueled an interest in elucidating the mechanisms of repair of interstrand cross-links (ICLs) 2The abbreviations used are: ICLinterstrand cross-linkNERnucleotide excision repairRPAreplication protein APCNAproliferating cell nuclear antigenWSWerner syndromesiRNAsmall interfering RNAGFPgreen fluorescent proteinGSTglutathione S-transferaseCRScross-link repair synthesis. that still remain largely unresolved in eukaryotic cells. In prokaryotes two mechanisms of repair of these lesions have been identified. A major error-free pathway involves incision by the nucleotide excision repair (NER) pathway and a subsequent recombination gap filling step mediated by RecA (1Cole R.S. Yale J. Biol. Med. 1973; 46: 492PubMed Google Scholar, 2Cole R.S. Levitan D. Sinden R.R. J. Mol. Biol. 1976; 103: 39-59Crossref PubMed Scopus (151) Google Scholar, 3Sladek F.M. Munn M.M. Rupp W.D. Howard-Flanders P. J. Biol. Chem. 1989; 264: 6755-6765Abstract Full Text PDF PubMed Google Scholar). A minor error-prone pathway also requires incision by NER, while the gap filling step is accomplished via translesion synthesis performed by DNA polymerase II (4Berardini M. Foster P.L. Loechler E.L. J. Bacteriol. 1999; 181: 2878-2882Crossref PubMed Google Scholar, 5Berardini M. Mackay W. Loechler E.L. Biochemistry. 1997; 36: 3506-3513Crossref PubMed Scopus (57) Google Scholar). In each case the remaining monoadduct is removed by a second round of NER action. In budding yeast there appears to be a general conservation of these pathways in that a major mechanism involving NER and recombination has been identified (6Jachymczyk W.J. von Borstel R.C. Mowat M.R. Hastings P.J. Mol. Gen. Genet. 1981; 182: 196-205Crossref PubMed Scopus (158) Google Scholar, 7Miller R.D. Prakash L. Prakash S. Mol. Cell. Biol. 1982; 2: 939-948Crossref PubMed Scopus (91) Google Scholar, 8Greenberg R.B. Alberti M. Hearst J.E. Chua M.A. Saffran W.A. J. Biol. Chem. 2001; 276: 31551-31560Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 9McHugh P.J. Gill R.D. Waters R. Hartley J.A. Nucleic Acids Res. 1999; 27: 3259-3266Crossref PubMed Scopus (55) Google Scholar), as well as a pathway involving the translesion polymerase Rev3 whose primary importance appears to occur in nonreplicating cells (9McHugh P.J. Gill R.D. Waters R. Hartley J.A. Nucleic Acids Res. 1999; 27: 3259-3266Crossref PubMed Scopus (55) Google Scholar). In all eukaryotic cells examined double-strand breaks have been observed as intermediates of ICL repair (10Akkari Y.M. Bateman R.L. Reifsteck C.A. Olson S.B. Grompe M. Mol. Cell. Biol. 2000; 20: 8283-8289Crossref PubMed Scopus (173) Google Scholar, 11Dardalhon M. Averbeck D. Mutat. Res. 1995; 336: 49-60Crossref PubMed Scopus (49) Google Scholar, 12De Silva I.U. McHugh P.J. Clingen P.H. Hartley J.A. Mol. Cell. Biol. 2000; 20: 7980-7990Crossref PubMed Scopus (387) Google Scholar, 13McHugh P.J. Sones W.R. Hartley J.A. Mol. Cell. Biol. 2000; 20: 3425-3433Crossref PubMed Scopus (139) Google Scholar) suggesting the possibility that recombination mechanisms may be required at more than one stage of this repair process. Mammalian cells have also been shown to employ two pathways of ICL repair. The minor pathway appears analogous to the Escherichia coli pathway in that it requires NER and presumably a translesion polymerase but not components of homologous recombination (14Wang X. Peterson C.A. Zheng H. Nairn R.S. Legerski R.J. Li L. Mol. Cell. Biol. 2001; 21: 713-720Crossref PubMed Scopus (125) Google Scholar, 15Zheng H. Wang X. Warren A.J. Legerski R.J. Nairn R.S. Hamilton J.W. Li L. Mol. Cell. Biol. 2003; 23: 754-761Crossref PubMed Scopus (131) Google Scholar). This pathway has also proved to be highly mutagenic in response to psoralen or mitomycin C adducts. The presumptive major pathway clearly requires homologous recombination, since mutations in genes such as XRCC1, XRCC2, and RAD51C result in severe sensitivity to ICL-inducing agents (16Liu N. Lamerdin J.E. Tebbs R.S. Schild D. Tucker J.D. Shen M.R. Brookman K.W. Siciliano M.J. Walter C.A. Fan W. Narayana L.S. Zhou Z.Q. Adamson A.W. Sorensen K.J. Chen D.J. Jones N.J. Thompson L.H. Mol. Cell. 1998; 1: 783-793Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar, 17Godthelp B.C. Wiegant W.W. van Duijn-Goedhart A. Scharer O.D. van Buul P.P. Kanaar R. Zdzienicka M.Z. Nucleic Acids Res. 2002; 30: 2172-2182Crossref PubMed Scopus (132) Google Scholar). Mutants in the NER genes ERCC1 and XPF are also strikingly sensitive to these drugs; however, somewhat surprisingly other components of the NER pathway exhibit only mild sensitivity and have been shown not to be defective in the unhooking step of repair of nitrogen mustard ICLs in vivo (12De Silva I.U. McHugh P.J. Clingen P.H. Hartley J.A. Mol. Cell. Biol. 2000; 20: 7980-7990Crossref PubMed Scopus (387) Google Scholar). In general, cellular genetic studies have not been particularly revealing about the early stages of the major pathway of ICL repair in mammalian cells. Our prior biochemical studies have indicated a requirement for the mismatch repair factor MutSβ, the heterodimer Ercc1-Xpf, and replication protein A (RPA) in early recognition and uncoupling of psoralen ICLs (18Li L. Peterson C.A. Lu X. Wei P. Legerski R.J. Mol. Cell. Biol. 1999; 19: 5619-5630Crossref PubMed Scopus (63) Google Scholar, 19Zhang N. Lu X. Zhang X. Peterson C.A. Legerski R.J. Mol. Cell. Biol. 2002; 22: 2388-2397Crossref PubMed Scopus (95) Google Scholar, 20Zhang N. Lu X. Legerski R.J. Biochem. Biophys. Res. Commun. 2003; 309: 71-78Crossref PubMed Scopus (19) Google Scholar, 21Zhang N. Zhang X. Peterson C. Li L. Legerski R. Nucleic Acids Res. 2000; 28: 4800-4804Crossref PubMed Scopus (24) Google Scholar). We have also shown that the recognition step by MutSβ is stimulated by PCNA. Consistent with these biochemical findings, it has been shown that Msh2 and Ercc1-Xpf act cooperatively in human cells to remove cisplatin ICLs and that these proteins interact as determined by co-immunoprecipitation experiments (22Lan L. Hayashi T. Rabeya R.M. Nakajima S. Kanno S. Takao M. Matsunaga T. Yoshino M. Ichikawa M. Riele H. Tsuchiya S. Tanaka K. Yasui A. DNA Repair (Amst.). 2004; 3: 135-143Crossref PubMed Scopus (55) Google Scholar). Furthermore, recent findings in budding yeast have implicated a role for Msh2 and Exo I in repair of nitrogen mustard ICLs (23Barber L.J. Ward T.A. Hartley J.A. McHugh P.J. Mol. Cell. Biol. 2005; 25: 2297-2309Crossref PubMed Scopus (61) Google Scholar). Taken together these findings suggest that mismatch repair factors and Ercc1-Xpf are involved in a novel pathway of ICL repair. interstrand cross-link nucleotide excision repair replication protein A proliferating cell nuclear antigen Werner syndrome small interfering RNA green fluorescent protein glutathione S-transferase cross-link repair synthesis. A number of genetic screens have been undertaken in budding yeast to identify novel genes involved in ICL repair. One isolated mutant, pso4-1, exhibited some sensitivity to irradiation but was particularly sensitive to psoralen and other ICL-inducing agents (24Henriques J.A. Vicente E.J. Leandro da Silva K.V. Schenberg A.C. Mutat. 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A. 2003; 100: 10746-10751Crossref PubMed Scopus (90) Google Scholar). Pso4/Prp19 interacts with other components of the spliceosome and forms a highly stable core complex with a number of proteins including Cdc5L (Cef1 in Saccharomyces cerevisiae) (28Ajuh P. Kuster B. Panov K. Zomerdijk J.C. Mann M. Lamond A.I. EMBO J. 2000; 19: 6569-6581Crossref PubMed Scopus (171) Google Scholar, 33Ohi M.D. Vander Kooi C.W. Rosenberg J.A. Ren L. Hirsch J.P. Chazin W.J. Walz T. Gould K.L. Mol. Cell. Biol. 2005; 25: 451-460Crossref PubMed Scopus (72) Google Scholar). WRN is a member of the RecQ family of DNA helicases and is mutated in the human autosomal recessive disease Werner syndrome (WS) (34Yu C.E. Oshima J. Fu Y.H. Wijsman E.M. Hisama F. Alisch R. Matthews S. Nakura J. Miki T. Ouais S. Martin G.M. Mulligan J. Schellenberg G.D. Science. 1996; 272: 258-262Crossref PubMed Scopus (1496) Google Scholar). 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Emond M.J. Monnat Jr., R.J. DNA Repair (Amst.). 2004; 3: 475-482Crossref PubMed Scopus (52) Google Scholar, 44Poot M. Yom J.S. Whang S.H. Kato J.T. Gollahon K.A. Rabinovitch P.S. FASEB J. 2001; 15: 1224-1226Crossref PubMed Scopus (133) Google Scholar, 45Poot M. Gollahon K.A. Emond M.J. Silber J.R. Rabinovitch P.S. FASEB J. 2002; 16: 757-758Crossref PubMed Scopus (84) Google Scholar). To investigate the mechanisms of ICL repair we have developed an in vitro assay in which repair processing in a plasmid substrate containing a site-specific psoralen cross-link can be monitored in mammalian cell extracts (18Li L. Peterson C.A. Lu X. Wei P. Legerski R.J. Mol. Cell. Biol. 1999; 19: 5619-5630Crossref PubMed Scopus (63) Google Scholar, 19Zhang N. Lu X. Zhang X. Peterson C.A. Legerski R.J. Mol. Cell. Biol. 2002; 22: 2388-2397Crossref PubMed Scopus (95) Google Scholar, 20Zhang N. Lu X. Legerski R.J. Biochem. Biophys. Res. Commun. 2003; 309: 71-78Crossref PubMed Scopus (19) Google Scholar, 21Zhang N. Zhang X. Peterson C. Li L. Legerski R. Nucleic Acids Res. 2000; 28: 4800-4804Crossref PubMed Scopus (24) Google Scholar, 46Li L. Peterson C.A. Zhang X. Legerski R.J. Nucleic Acids Res. 2000; 28: 1424-1427Crossref PubMed Scopus (26) Google Scholar). Using this assay, we report here the fractionation and identification of a protein complex composed of Pso4/Prp19, Cdc5L, Plrg1, and Spf27 (Pso4 complex) that is required for processing of psoralen ICLs in vitro. In addition, we show that the WRN protein is also absolutely essential to this processing and that WRN interacts with the Pso4 complex mediated by a direct association with Cdc5L. Cell Culture—Human lymphoid cell lines were cultured in suspension in RPMI 1640 medium with 20% fetal calf serum. HeLa cells and HEK293 cells were cultured in DMEM containing 10% serum. Transfection of plasmids and siRNA were performed using FuGENE 6 and Oligofectamine (Invitrogen), respectively, following the manufacturer's instructions. Cells were harvested 48 h after the transfection and prepared for co-immunoprecipitation or colonogenesis assays. Recombinant Proteins and Antibodies— cDNAs for the Pso4 complex proteins were cloned into the Gateway cloning system (Invitrogen) for expression in either E. coli, SF9 insect cells, or mammalian cells. The vectors expressing WRN proteins were generously provided by Judith Campisi. Recombinant 6×His-Cdc5L, 6×His-Plrg1, 6×His-Pso4, 6×His-Spf27, 6×His-WRN, 6×His-WRN(K84A), and 6×His-WRN-(1-333) were expressed in Sf9 insect cells using a baculovirus expression system. Proteins were purified by nickel affinity and Mono Q chromatography. Polyclonal antibodies against Cdc5L, Plrg1, Pso4, and Spf27 were generously provided by Paul Ajuh (28Ajuh P. Kuster B. Panov K. Zomerdijk J.C. Mann M. Lamond A.I. EMBO J. 2000; 19: 6569-6581Crossref PubMed Scopus (171) Google Scholar). A monoclonal antibody against Cdc5L was purchased from BD Biosciences. Monoclonal antibodies against GST and GFP are from Santa Cruz Biotechnology. Polyclonal and monoclonal antibodies against WRN was purchased from Abcam and Sigma, respectively. Fractionation of HeLa Nuclear Extract and in Vitro Interstrand Cross-link Repair (CRS) Assay—HeLa nuclear extracts were prepared as described previously (20Zhang N. Lu X. Legerski R.J. Biochem. Biophys. Res. Commun. 2003; 309: 71-78Crossref PubMed Scopus (19) Google Scholar, 47Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9164) Google Scholar). Nuclear extract was subjected to sequential chromatography using P11 phosphocellulose, hydroxyapatite, heparin-agarose, Mono S, and Mono Q. Active fractions were determined by reconstitution of the CRS assay with fractions FI and FII. The active fraction from the Mono Q column was applied to 12% SDS-PAGE, protein bands were visualized by silver staining, and isolated bands were subjected to mass spectrography analysis. Psoralen interstrand cross-linked substrates for the CRS assay were prepared as described previously (18Li L. Peterson C.A. Lu X. Wei P. Legerski R.J. Mol. Cell. Biol. 1999; 19: 5619-5630Crossref PubMed Scopus (63) Google Scholar). Mammalian whole-cell extracts were prepared as described (48Manley J.L. Fire A. Cano A. Sharp P.A. Gefter M.L. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3855-3859Crossref PubMed Scopus (735) Google Scholar) and were tested for competency in an in vitro NER assay (49Wood R.D. Robins P. Lindahl T. Cell. 1988; 53: 97-106Abstract Full Text PDF PubMed Scopus (380) Google Scholar). The CRS assay was performed as described previously (18Li L. Peterson C.A. Lu X. Wei P. Legerski R.J. Mol. Cell. Biol. 1999; 19: 5619-5630Crossref PubMed Scopus (63) Google Scholar, 19Zhang N. Lu X. Zhang X. Peterson C.A. Legerski R.J. Mol. Cell. Biol. 2002; 22: 2388-2397Crossref PubMed Scopus (95) Google Scholar). For reconstitution assays, 25 μg of HeLa nuclear extract, or 10 μg of fraction FI, 10 μg of fraction FII, and 2-4 μl of fraction FIII or the chromatographic fractions derived from FIII were used for each reaction. Immunodepletion, Co-immunoprecipitation, and Pull-down Assays—Immunodepletion and coimmunoprecipitation assays were performed as described previously (50Zhang X. Succi J. Feng Z. Prithivirajsingh S. Story M.D. Legerski R.J. Mol. Cell. Biol. 2004; 24: 9207-9220Crossref PubMed Scopus (101) Google Scholar). For pull-down assays GST-tagged proteins were bound on glutathione-Sepharose beads according to manufacturer's instructions (Amersham Biosciences). The immobilized proteins were washed with Nonidet P-40 buffer (20 mm Tris, pH 8, 100 mm NaCl, 1 mm EDTA, 0.5% Nonidet P-40), then nuclear extract or purified proteins was added to the beads and incubated for 2 h at 4°C. The unbound proteins were removed by washing the beads four times with 100 bed volumes of Nonidet P-40 buffer. The bound proteins were eluted by boiling in SDS-PAGE sample buffer and visualized after gel electrophoresis by immunoblot analysis. Colonogenic Survival Assay—Cdc5L and Pso4 were depleted by transfection of HeLa cells with siRNAs. The siRNA sequences used to knockdown Cdc5L and Pso4 were GGAAGAGAGGAGUUGAUUA and ACCACAGGCUGGCCUCAUU, respectively. Forty-eight hours after transfection, cells were trypsinized and plated at the indicated density. Twelve to 15 days later, colonies were fixed and stained with crystal violet (0.25% crystal violet, 0.2% paraformaldehyde, 72% methanol, 18% water) and counted. Reactivation Assay—Substrate preparation and the luciferase-based reactivation were performed as described previously (15Zheng H. Wang X. Warren A.J. Legerski R.J. Nairn R.S. Hamilton J.W. Li L. Mol. Cell. Biol. 2003; 23: 754-761Crossref PubMed Scopus (131) Google Scholar). The Pso4 Complex Is Required for in Vitro Processing of ICLs—To elucidate the processing of ICLs in mammalian cells, we have developed an in vitro assay (referred to as CRS) in which a single psoralen ICL located at a defined site within a plasmid substrate induces DNA synthesis in both damaged and undamaged plasmids in cell-free extracts (18Li L. Peterson C.A. Lu X. Wei P. Legerski R.J. Mol. Cell. Biol. 1999; 19: 5619-5630Crossref PubMed Scopus (63) Google Scholar). The substrates for this assay are described in the legend to Fig. 1A. The donor template plasmid is added to the reactions primarily as a carrier to absorb nonspecific inhibitory DNA binding proteins. As shown previously (19Zhang N. Lu X. Zhang X. Peterson C.A. Legerski R.J. Mol. Cell. Biol. 2002; 22: 2388-2397Crossref PubMed Scopus (95) Google Scholar), the products of the in vitro processing of psoralen ICLs are two oligonucleotides migrating at the positions of 113 and 86 nucleotides on denaturing gels, which are derived from uncoupling of the ICL (supplemental Fig. S1). To identify additional factors involved in the processing of ICLs, we have initiated fractionation of HeLa nuclear extracts. P11 phosphocellulose column chromatography of HeLa nuclear extracts yielded four fractions, FI-FIV, the first three of which are required to reconstitute the assay, while FIV is dispensable (Fig. 1B) (20Zhang N. Lu X. Legerski R.J. Biochem. Biophys. Res. Commun. 2003; 309: 71-78Crossref PubMed Scopus (19) Google Scholar). RPA and PCNA can substitute for fraction FI, and Ercc1-Xpf and MutSβ are located in fraction FII but are not sufficient to substitute for this fraction (20Zhang N. Lu X. Legerski R.J. Biochem. Biophys. Res. Commun. 2003; 309: 71-78Crossref PubMed Scopus (19) Google Scholar). The factor(s) present in FIII were unknown, and thus we undertook the purification of these factor(s) using complementation of fractions FI and FII in the CRS assay. After five chromatographic steps (phosphocellulose, heparin, hydroxyapatite, Mono S, Mono Q) a single active fraction was obtained from the Mono Q column, and the bands detected by silver staining were processed for identification by mass spectroscopy (Fig. 1C). This analysis identified a complex containing the human homolog of the S. cerevisiae protein Pso4/Prp19 and three other proteins, the human homolog of Schizosaccharomyces pombe Cdc5 (Cdc5L), Plrg1, and Spf27 (Fig. 1D, upper panel). The identity of all four of these proteins was verified by Western blotting, and the Cdc5L and Pso4 results are shown (Fig. 1D, lower panel). All four of these proteins have been shown previously to be core components of a human Cdc5L-containing sub-pre-mRNA spliceosome complex that is conserved from yeast to mammals (28Ajuh P. Kuster B. Panov K. Zomerdijk J.C. Mann M. Lamond A.I. EMBO J. 2000; 19: 6569-6581Crossref PubMed Scopus (171) Google Scholar, 30McDonald W.H. Ohi R. Smelkova N. Frendewey D. Gould K.L. Mol. Cell. Biol. 1999; 19: 5352-5362Crossref PubMed Scopus (111) Google Scholar, 51Burns C.G. Ohi R. Krainer A.R. Gould K.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13789-13794Crossref PubMed Scopus (63) Google Scholar, 52Neubauer G. King A. Rappsilber J. Calvio C. Watson M. Ajuh P. Sleeman J. Lamond A. Mann M. Nat. Genet. 1998; 20: 46-50Crossref PubMed Scopus (421) Google Scholar, 53Tsai W.Y. Chow Y.T. Chen H.R. Huang K.T. Hong R.I. Jan S.P. Kuo N.Y. Tsao T.Y. Chen C.H. Cheng S.C. J. Biol. Chem. 1999; 274: 9455-9462Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Interestingly, the only known catalytic domain in any of the four proteins is an ubiquitin E3 ligase located in the U-box domain of Pso4/Prp19 (54Hatakeyama S. Yada M. Matsumoto M. Ishida N. Nakayama K.I. J. Biol. Chem. 2001; 276: 33111-33120Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar, 55Ohi M.D. Vander Kooi C.W. Rosenberg J.A. Chazin W.J. Gould K.L. Nat. Struct. Biol. 2003; 10: 250-255Crossref PubMed Scopus (229) Google Scholar, 56Loscher M. Fortschegger K. Ritter G. Wostry M. Voglauer R. Schmid J.A. Watters S. Rivett A.J. Ajuh P. Lamond A.I. Katinger H. Grillari J. Biochem. J. 2005; 388: 593-603Crossref PubMed Scopus (52) Google Scholar). In addition, two of the proteins, Pso4 and Plrg1, contain WD40 domains, all of which suggests that these proteins may play a largely structural role. For convenience, we will here designate these four proteins as the Pso4 complex. To verify the role of the Pso4 complex in ICL processing, we first immunodepleted Cdc5L from HeLa extracts (Fig. 2A) and then examined the activity of these depleted extracts in the CRS assay. As shown (Fig. 2B), depletion of Cdc5L resulted in a substantial reduction in the CRS assay. Furthermore, addition of purified recombinant Pso4 complex prepared from baculovirus (shown in Fig. 2C) was able to rescue the Cdc5L-depleted extracts. Next we examined various combinations of the recombinant Pso4 complex in the CRS assay. As shown (Fig. 2D, left panel), none of the individual proteins could substitute for FIII; however, addition of all four proteins did result in complementation approximately equivalent to that observed upon addition of FIII. In addition, no combination of any three of the proteins was able to effect complementation in the assay (Fig. 2D, right panel). Thus, we conclude that all four proteins are required for processing of psoralen ICLs in vitro and that the Pso4 complex is sufficient to substitute for fraction FIII in the CRS assay. Pso4 and Cdc5L Are Involved in Repair of ICLs in Vivo—To determine whether Pso4/Prp19 or Cdc5L is involved in mediating resistance to ICL-inducing agents in vivo, we used siRNA to deplete these proteins in HeLa cells. However, in contrast to a previous report (32Mahajan K.N. Mitchell B.S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10746-10751Crossref PubMed Scopus (90) Google Scholar) we found that transfection of duplex siRNA oligonucleotides targeting either PSO4 or CDC5L was growth inhibitory for HeLa cells (supplemental Fig. S2), thus precluding cell survival studies after exposure to genotoxic agents. The reason for this discrepancy is not clear, but both of these factors are essential in yeast cells due to their role in pre-mRNA splicing, and this role appears to be conserved in mammalian cells (28Ajuh P. Kuster B. Panov K. Zomerdijk J.C. Mann M. Lamond A.I. EMBO J. 2000; 19: 6569-6581Crossref PubMed Scopus (171) Google Scholar, 57Ajuh P. Lamond A.I. Nucleic Acids Res. 2003; 31: 6104-6116Crossref PubMed Scopus (21) Google Scholar, 58Jurica M.S. Moore M.J. Mol. Cell. 2003; 12: 5-14Abstract Full Text Full Text PDF PubMed Scopus (797) Google Scholar). We have shown in recent studies that wild-type mammalian cells are able to repair cross-linked plasmids upon transfection as determined by a luciferase reactivation assay (14Wang X. Peterson C.A. Zheng H. Nairn R.S. Legerski R.J. Li L. Mol. Cell. Biol. 2001; 21: 713-720Crossref PubMed Scopus (125) Google Scholar, 15Zheng H. Wang X. Warren A.J. Legerski R.J. Nairn R.S. Hamilton J.W. Li L. Mol. Cell. Biol. 2003; 23: 754-761Crossref PubMed Scopus (131) Google Scholar). We therefore used this short term assay after knockdown of Pso4 or Cdc5L by siRNA to examine the role of these factors in ICL repair in vivo. As shown (Fig. 3), depletion of either Pso4 or Cdc5L showed a substantial and highly reproducible reduction in the reactivation of psoralen cross-linked plasmids. These results were not due to differences in cellular transfection efficiency, since a GFP-expressing plasmid was included in each experiment to allow for normalization of the luciferase reactivation. The Pso4 Complex Associates with the Werner Syndrome Protein through a Direct Interaction with Cdc5L—To determine whether the Pso4 complex interacted with the previously identified components of in vitro ICL processing, we performed co-immunoprecipitation experiments. Antibodies to either Cdc5L or Pso4 were used for immunoprecipitation, and blots were subsequently analyzed for the presence of Msh2, Xpf, and the middle subunit of RPA; however, none of these proteins were detected (results not shown). Previous studies have shown that Werner syndrome cell lines are highly sensit
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