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• W1992129085 abstract "The endonuclease ERCC1-XPF incises the damaged strand of DNA 5′ to a lesion during nucleotide excision repair (NER) and has additional, poorly characterized functions in interstrand cross-link repair, double-strand break repair, and homologous recombination. XPA, another key factor in NER, interacts with ERCC1 and recruits it to sites of damage. We identified ERCC1 residues that are critical for the interaction with XPA and assessed their importance for NER in vitro and in vivo. Mutation of two conserved residues (Asn-110 and Tyr-145) located in the XPA-binding site of ERCC1 dramatically affected NER but not nuclease activity on model DNA substrates. In ERCC1-deficient cells expressing ERCC1N110A/Y145A, the nuclease was not recruited to sites of UV damage. The repair of UV-induced (6-4)photoproducts was severely impaired in these cells, and they were hypersensitive to UV irradiation. Remarkably, the ERCC1N110A/Y145A protein rescues the sensitivity of ERCC1-deficient cells to cross-linking agents. Our studies suggest that ERCC1-XPF engages in different repair pathways through specific protein-protein interactions and that these functions can be separated through the selective disruption of these interactions. We discuss the impact of these findings for understanding how ERCC1 contributes to resistance of tumor cells to therapeutic agents such as cisplatin. The endonuclease ERCC1-XPF incises the damaged strand of DNA 5′ to a lesion during nucleotide excision repair (NER) and has additional, poorly characterized functions in interstrand cross-link repair, double-strand break repair, and homologous recombination. XPA, another key factor in NER, interacts with ERCC1 and recruits it to sites of damage. We identified ERCC1 residues that are critical for the interaction with XPA and assessed their importance for NER in vitro and in vivo. Mutation of two conserved residues (Asn-110 and Tyr-145) located in the XPA-binding site of ERCC1 dramatically affected NER but not nuclease activity on model DNA substrates. In ERCC1-deficient cells expressing ERCC1N110A/Y145A, the nuclease was not recruited to sites of UV damage. The repair of UV-induced (6-4)photoproducts was severely impaired in these cells, and they were hypersensitive to UV irradiation. Remarkably, the ERCC1N110A/Y145A protein rescues the sensitivity of ERCC1-deficient cells to cross-linking agents. Our studies suggest that ERCC1-XPF engages in different repair pathways through specific protein-protein interactions and that these functions can be separated through the selective disruption of these interactions. We discuss the impact of these findings for understanding how ERCC1 contributes to resistance of tumor cells to therapeutic agents such as cisplatin. IntroductionThe preservation of the genetic information contained in DNA is essential for proper cell function and is ensured by multiple DNA repair pathways. Among these, nucleotide excision repair (NER) 2The abbreviations used are: NERnucleotide excision repairICLinterstrand cross-linkXPxeroderma pigmentosumERCCexcision repair cross-complementing(6-4)PP(6-4)photoproductCHOChinese hamster ovaryPBSphosphate-buffered salineDSBdouble strand breakWTwild type. clears the genome of bulky, helix-distorting DNA lesions, such as those formed by UV light, environmental mutagens, and antitumor agents (1Friedberg E.C. Walker G.C. Siede W. Wood R.D. Schultz R.A. Ellenberger T. DNA Repair and Mutagenesis. 2nd Ed. ASM Press, Washington, D. C.2005Crossref Google Scholar, 2Gillet L.C. Schärer O.D. Chem. Rev. 2006; 106: 253-276Crossref PubMed Scopus (488) Google Scholar). Two subpathways of NER exist that differ in their method of damage recognition. In transcription-coupled NER, lesions in the transcribed strand of genes block the progression of RNA polymerase II, triggering NER (3Hanawalt P.C. Spivak G. Nat. Rev. Mol. Cell Biol. 2008; 9: 958-970Crossref PubMed Scopus (761) Google Scholar). In global genome NER, helix-distorting lesions anywhere in the genome are recognized by the XPC-RAD23B heterodimer (4Sugasawa K. Ng J.M. Masutani C. Iwai S. van der Spek P.J. Eker A.P. Hanaoka F. Bootsma D. Hoeijmakers J.H. Mol. Cell. 1998; 2: 223-232Abstract Full Text Full Text PDF PubMed Scopus (741) Google Scholar), in some cases with the help of UV-DDB (5Sugasawa K. Okuda Y. Saijo M. Nishi R. Matsuda N. Chu G. Mori T. Iwai S. Tanaka K. Tanaka K. Hanaoka F. Cell. 2005; 121: 387-400Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar). The subsequent steps of NER are believed to be similar for both subpathways and occur by the sequential assembly of the NER proteins at the site of the lesion (6Volker M. Moné M.J. Karmakar P. van Hoffen A. Schul W. Vermeulen W. Hoeijmakers J.H. van Driel R. van Zeeland A.A. Mullenders L.H. Mol. Cell. 2001; 8: 213-224Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar, 7Riedl T. Hanaoka F. Egly J.M. EMBO J. 2003; 22: 5293-5303Crossref PubMed Scopus (342) Google Scholar, 8Fousteri M. Vermeulen W. van Zeeland A.A. Mullenders L.H. Mol. Cell. 2006; 23: 471-482Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). Recruitment of TFIIH containing two helicase subunits XPB and XPD leads to the separation of the damaged and undamaged DNA strands (9Evans E. Moggs J.G. Hwang J.R. Egly J.M. Wood R.D. EMBO J. 1997; 16: 6559-6573Crossref PubMed Scopus (398) Google Scholar, 10Tapias A. Auriol J. Forget D. Enzlin J.H. Schärer O.D. Coin F. Coulombe B. Egly J.M. J. Biol. Chem. 2004; 279: 19074-19083Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). This enables subsequent NER factors to bind, including XPA, the single-stranded binding protein RPA, and the endonuclease XPG (7Riedl T. Hanaoka F. Egly J.M. EMBO J. 2003; 22: 5293-5303Crossref PubMed Scopus (342) Google Scholar). The last factor to be recruited to this preincision complex is the endonuclease ERCC1-XPF (6Volker M. Moné M.J. Karmakar P. van Hoffen A. Schul W. Vermeulen W. Hoeijmakers J.H. van Driel R. van Zeeland A.A. Mullenders L.H. Mol. Cell. 2001; 8: 213-224Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar, 11Wakasugi M. Sancar A. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 6669-6674Crossref PubMed Scopus (149) Google Scholar). Once ERCC1-XPF is properly positioned on the DNA, via its interactions with XPA and RPA (12de Laat W.L. Appeldoorn E. Sugasawa K. Weterings E. Jaspers N.G. Hoeijmakers J.H. Genes Dev. 1998; 12: 2598-2609Crossref PubMed Scopus (260) Google Scholar, 13Camenisch U. Dip R. Schumacher S.B. Schuler B. Naegeli H. Nat. Struct. Mol. Biol. 2006; 13: 278-284Crossref PubMed Scopus (84) Google Scholar), it incises the damage strand 5′ to the lesion followed by XPG making the 3′ incision (14Staresincic L. Fagbemi A.F. Enzlin J.H. Gourdin A.M. Wijgers N. Dunand-Sauthier I. Giglia-Mari G. Clarkson S.G. Vermeulen W. Schärer O.D. EMBO J. 2009; 28: 1111-1120Crossref PubMed Scopus (183) Google Scholar), allowing the replicative DNA polymerases and associated factors to fill the gap and restore the original DNA sequence (15Mocquet V. Lainé J.P. Riedl T. Yajin Z. Lee M.Y. Egly J.M. EMBO J. 2008; 27: 155-167Crossref PubMed Scopus (127) Google Scholar, 16Ogi T. Lehmann A.R. Nat. Cell Biol. 2006; 8: 640-642Crossref PubMed Scopus (128) Google Scholar, 17Moser J. Kool H. Giakzidis I. Caldecott K. Mullenders L.H. Fousteri M.I. Mol. Cell. 2007; 27: 311-323Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar).Inherited defects in NER cause xeroderma pigmentosum (XP) characterized by extreme photosensitivity and risk of skin cancer (1Friedberg E.C. Walker G.C. Siede W. Wood R.D. Schultz R.A. Ellenberger T. DNA Repair and Mutagenesis. 2nd Ed. ASM Press, Washington, D. C.2005Crossref Google Scholar). Genetic defects in NER factors underlie additional disorders. Mutations in XPB, XPD, TTD-A, and XPG (all associated with TFIIH) can cause Cockayne syndrome, combined XP/Cockayne syndrome, or trichothiodystrophy, characterized by severe developmental defects and neurodegeneration, due to the additional role of these gene products in transcription (18Lehmann A.R. Biochimie. 2003; 85: 1101-1111Crossref PubMed Scopus (401) Google Scholar). Mutations in ERCC1 cause cerebro-oculo-facio-skeletal syndrome, whereas mutations in XPF that severely affect protein expression cause accelerated aging (XFE progeroid syndrome (19Niedernhofer L.J. Garinis G.A. Raams A. Lalai A.S. Robinson A.R. Appeldoorn E. Odijk H. Oostendorp R. Ahmad A. van Leeuwen W. Theil A.F. Vermeulen W. van der Horst G.T. Meinecke P. Kleijer W.J. Vijg J. Jaspers N.G. Hoeijmakers J.H. Nature. 2006; 444: 1038-1043Crossref PubMed Scopus (521) Google Scholar, 20Jaspers N.G. Raams A. Silengo M.C. Wijgers N. Niedernhofer L.J. Robinson A.R. Giglia-Mari G. Hoogstraten D. Kleijer W.J. Hoeijmakers J.H. Vermeulen W. Am. J. Hum. Genet. 2007; 80: 457-466Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar)). Mice completely deficient in ERCC1-XPF also age rapidly and have a dramatically reduced life span (21McWhir J. Selfridge J. Harrison D.J. Squires S. Melton D.W. Nat. Genet. 1993; 5: 217-224Crossref PubMed Scopus (279) Google Scholar, 22Weeda G. Donker I. de Wit J. Morreau H. Janssens R. Vissers C.J. Nigg A. van Steeg H. Bootsma D. Hoeijmakers J.H. Curr. Biol. 1997; 7: 427-439Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 23Tian M. Shinkura R. Shinkura N. Alt F.W. Mol. Cell. Biol. 2004; 24: 1200-1205Crossref PubMed Scopus (130) Google Scholar). The severe phenotype caused by deficiency of ERCC1-XPF, relative to XP, has been ascribed to the additional role of this NER factor in the repair of interstrand cross-links (ICLs) (19Niedernhofer L.J. Garinis G.A. Raams A. Lalai A.S. Robinson A.R. Appeldoorn E. Odijk H. Oostendorp R. Ahmad A. van Leeuwen W. Theil A.F. Vermeulen W. van der Horst G.T. Meinecke P. Kleijer W.J. Vijg J. Jaspers N.G. Hoeijmakers J.H. Nature. 2006; 444: 1038-1043Crossref PubMed Scopus (521) Google Scholar).The recruitment of ERCC1-XPF to various DNA repair pathways is predicted to be mediated through specific protein-protein interactions. The interaction between XPA and ERCC1 is essential for NER involving a region of XPA encompassing three consecutive glycines (residues 72–74) (24Park C.H. Sancar A. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 5017-5021Crossref PubMed Scopus (163) Google Scholar, 25Li L. Elledge S.J. Peterson C.A. Bales E.S. Legerski R.J. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 5012-5016Crossref PubMed Scopus (270) Google Scholar, 26Li L. Peterson C.A. Lu X. Legerski R.J. Mol. Cell. Biol. 1995; 15: 1993-1998Crossref PubMed Scopus (113) Google Scholar). The structural basis for the interaction between XPA and ERCC1 was recently established (27Tsodikov O.V. Ivanov D. Orelli B. Staresincic L. Shoshani I. Oberman R. Schärer O.D. Wagner G. Ellenberger T. EMBO J. 2007; 26: 4768-4776Crossref PubMed Scopus (114) Google Scholar, 28Tripsianes K. Folkers G.E. Zheng C. Das D. Grinstead J.S. Kaptein R. Boelens R. Nucleic Acids Res. 2007; 35: 5789-5798Crossref PubMed Scopus (37) Google Scholar). A short and unstructured peptide of XPA, XPA67–80, including the three aforementioned glycine residues, undergoes a disorder-to-order transition upon binding to the central domain of ERCC1 (see Fig. 1). This 14- amino acid stretch of XPA is necessary and sufficient to mediate its interaction with ERCC1, and the XPA67–80 peptide can inhibit NER activity in vitro (27Tsodikov O.V. Ivanov D. Orelli B. Staresincic L. Shoshani I. Oberman R. Schärer O.D. Wagner G. Ellenberger T. EMBO J. 2007; 26: 4768-4776Crossref PubMed Scopus (114) Google Scholar).These findings prompted us to investigate how the XPA-binding region of ERCC1 contributes to NER activity and other DNA repair functions of ERCC1-XPF. Herein, we report mutations in the central domain of ERCC1 that severely impact NER activity in vitro and in vivo. Importantly, these mutations did not affect the ability of ERCC1-XPF to function in other DNA repair pathways. Our studies suggest that the roles of ERCC1-XPF can be separated by disrupting specific protein interactions that target the endonuclease to different DNA repair pathways.RESULTSA short polypeptide from XPA (residues 67–80) binds in a groove on the central domain of ERCC1 spanning residues 105–160. Mutations that alter the strictly conserved XPA residues Gly-72, Gly-73, Gly-74, or Phe-75 abolish NER activity in vitro (27Tsodikov O.V. Ivanov D. Orelli B. Staresincic L. Shoshani I. Oberman R. Schärer O.D. Wagner G. Ellenberger T. EMBO J. 2007; 26: 4768-4776Crossref PubMed Scopus (114) Google Scholar). To study the functional role of the XPA-binding groove of ERCC1 in NER and possibly ICL and DSB repair, we set out to design mutations in ERCC1 that would disrupt the interaction with XPA. Inspection of the XPA-binding site suggested that three absolutely conserved residues in ERCC1 might be important for this interaction (supplemental Fig. 1); ERCC1 residue Asn-110 packs against XPA residue Phe-75, ERCC1 Tyr-145 contacts XPA Gly-74, and ERCC1 Tyr-152 contacts XPA residues Gly-73 and Gly-72 (Fig. 1). These ERCC1 residues are located on three different aspects of the pocket that accepts the XPA peptide, and their side chains contribute a substantial portion of the binding surface. ERCC1 residues Asn-110, Tyr-145, and Tyr-152 were mutated to alanine using site-directed mutagenesis, and we additionally generated the double mutants N110A/Y145A and Y145A/Y152A.Mutations in the XPA-binding Domain of ERCC1 Affect NER but Not the Nuclease Activity of ERCC1-XPFThe mutant ERCC1 proteins were co-expressed with wild-type XPF in Sf9 insect cells, and the resulting heterodimers were purified in three chromatographic steps using nickel-nitrilotriacetic acid, size exclusion, and heparin columns (29Enzlin J.H. Schärer O.D. EMBO J. 2002; 21: 2045-2053Crossref PubMed Scopus (161) Google Scholar). All of the mutant ERCC1 proteins eluted from the size exclusion column as proper heterodimers (supplemental Fig. 2A), indicating that mutations in the XPA-binding domain of ERCC1 did not disrupt the protein fold of ERCC1 or its interaction with XPF. All the proteins were judged to be >95% pure (supplemental Fig. 2B).We first tested the effects of the ERCC1 N110A, Y145A, and Y152A mutations on the ability of ERCC1-XPF to incise DNA. The ERCC1 mutant protein complexes were incubated with a stem-loop DNA substrate in the presence of MnCl2 (34de Laat W.L. Appeldoorn E. Jaspers N.G. Hoeijmakers J.H. J. Biol. Chem. 1998; 273: 7835-7842Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar), and the release of a 10-mer oligonucleotide product was detected by denaturing PAGE analysis (Fig. 2A). ERCC1-XPF heterodimers containing mutant or wild-type ERCC1 subunits processed this model DNA substrate with similar efficiencies, cleaving at the single-stranded/double-stranded DNA junction of the stem loop. These results indicate that mutation of the Asn-110, Tyr-145, and Tyr-152 residues in ERCC1 does not affect the DNA binding and nuclease activities of the ERCC1-XPF heterodimer.FIGURE 2Mutations in the XPA-binding domain of ERCC1 affect NER activity but not nuclease activity in vitro. A, incision of a stem loop substrate by wild-type and mutant ERCC1-XPF. A 5′-32P-labeled stem-loop DNA substrate (6.7 nm) was incubated with 6.7 nm (lanes 2, 4, 6, 8, 10, and 12) or 26.8 nm (lanes 3, 5, 7, 9, 11, and 13) ERCC1-XPF in the presence of 0.4 mm MnCl2. B, NER activity of wild-type and mutant ERCC1-XPF. A plasmid containing a site-specific 1,3-intrastrand cisplatin DNA cross-link (50 ng) was incubated with a whole cell extract from ERCC1-XPF-deficient cells (XP2YO) complemented with recombinant ERCC1-XPF containing the indicated mutations in ERCC1 (N110A, Y145A, Y152A) or XPF (D720A). The excised DNA fragments of 24–32 nucleotides were detected by annealing a complementary oligonucleotide containing a non-complementary 4G overhang and filling in with [α-32P]dCTP. Protein concentrations of ERCC1-XPF were 13.4 nm (lanes 2, 4, 6, 8, 10, 12, and 14) and 53.6 nm (lanes 3, 5, 7, 9, 11, 13, and 15). A labeled low molecular weight DNA ladder (New England Biolabs) was used as a marker. The position of a 25-mer is indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We next tested the effects of these mutations on NER activity in cell-free extracts. A plasmid containing a site-specific 1,3-intrastrand cisplatin DNA cross-link was incubated with a cell-free extract from XPF-deficient cells (XP-F cells are devoid of both XPF and ERCC1 proteins (35Biggerstaff M. Szymkowski D.E. Wood R.D. EMBO J. 1993; 12: 3685-3692Crossref PubMed Scopus (147) Google Scholar, 36van Vuuren A.J. Appeldoorn E. Odijk H. Yasui A. Jaspers N.G. Bootsma D. Hoeijmakers J.H. EMBO J. 1993; 12: 3693-3701Crossref PubMed Scopus (140) Google Scholar, 37Yagi T. Wood R.D. Takebe H. Mutagenesis. 1997; 12: 41-44Crossref PubMed Scopus (43) Google Scholar)) that was complemented with recombinant ERCC1-XPF proteins containing wild-type or mutant ERCC1 subunits (30Shivji M.K. Moggs J.G. Kuraoka I. Wood R.D. Methods Mol. Biol. 1999; 113: 373-392PubMed Google Scholar). The unsupplemented XP-F cell extract lacked detectable NER activity (Fig. 2B, lane 1). The addition of ERCC1WT-XPF protein to the extract restored NER activity, generating the characteristic excision products of 24–32 nucleotides in length (Fig. 2B, lanes 2 and 3). All mutations in the XPA-binding site of ERCC1 led to a decrease in NER activity, with the strongest effects observed for the ERCC1N110A single, ERCC1Y145A/Y152A, and ERCC1N110A/Y145A double mutants (Fig. 2B, lanes 4–13). The activity of ERCC1N110A/Y145A was only slightly above the background level measured with the catalytically inactive mutant XPFD720A (14Staresincic L. Fagbemi A.F. Enzlin J.H. Gourdin A.M. Wijgers N. Dunand-Sauthier I. Giglia-Mari G. Clarkson S.G. Vermeulen W. Schärer O.D. EMBO J. 2009; 28: 1111-1120Crossref PubMed Scopus (183) Google Scholar) (Fig. 2B, lanes 14 and 15). These results demonstrate that an intact XPA-binding pocket in ERCC1 is required for NER activity in vitro.XPA-binding Mutants of ERCC1 Do Not Localize to Sites of UV DamageHaving established that the XPA-interaction mutants of ERCC1 are defective in NER in vitro, we assessed whether they prevent the recruitment of ERCC1-XPF to sites of UV damage in living cells. Based on the in vitro results (Fig. 2), we chose the ERCC1N110A, ERCC1Y145A, and ERCC1N110A/Y145A mutants for the cellular studies. ERCC1-deficient UV20 CHO cells (38Rolig R.L. Lowery M.P. Adair G.M. Nairn R.S. Mutagenesis. 1998; 13: 357-365Crossref PubMed Scopus (12) Google Scholar) were transduced with recombinant lentiviral vectors expressing mutant and wild-type ERCC1 cDNAs (14Staresincic L. Fagbemi A.F. Enzlin J.H. Gourdin A.M. Wijgers N. Dunand-Sauthier I. Giglia-Mari G. Clarkson S.G. Vermeulen W. Schärer O.D. EMBO J. 2009; 28: 1111-1120Crossref PubMed Scopus (183) Google Scholar, 31Dunand-Sauthier I. Hohl M. Thorel F. Jaquier-Gubler P. Clarkson S.G. Schärer O.D. J. Biol. Chem. 2005; 280: 7030-7037Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Immunoblotting revealed that the transduced UV20 cells stably expressed human ERCC1 protein (Fig. 3A).FIGURE 3Mutations in the XPA-binding domain of ERCC1 affect its recruitment to sites of UV damage. A, expression levels of ERCC1 in transduced UV20 cells. Transduced cells express human ERCC1 tagged with hemagglutinin. Note that human ERCC1 has a slower mobility than the CHO protein due to larger size (297 amino acids versus 293 amino acids) and the presence of the hemagglutinin tag. Tubulin was used as a loading control. B, ERCC1-deficient CHO cells were transduced with wild-type or mutant ERCC1 and irradiated with UV light (120 J/m2) through a polycarbonate filter with 5-μm pores and then fixed and stained for ERCC1 (green) and (6-4)PP (red). DAPI, 4′-6′-diamino-2-phenylindole. C, graphical representation of the percentage of co-localization of ERCC1 with (6-4)PP in UV20 cells expressing various mutants of ERCC1. Data represent the average of at least three independent experiments ± S.D. (error bars). 100 cells were counted for each experiment.View Large Image Figure ViewerDownload Hi-res image Download (PPT)UV20 cells expressing wild-type and mutant ERCC1 were UV-irradiated through a polycarbonate filter with 5-μm pores to generate a pattern of localized DNA damage that could be visualized by immunofluorescence microscopy (6Volker M. Moné M.J. Karmakar P. van Hoffen A. Schul W. Vermeulen W. Hoeijmakers J.H. van Driel R. van Zeeland A.A. Mullenders L.H. Mol. Cell. 2001; 8: 213-224Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar). 1 h after UV irradiation, the cells were fixed, and the presence of (6-4)photoproducts ((6-4)PP) and of ERCC1 at sites of UV damage was monitored by immunofluorescence. As expected, UV20 cells expressing wild-type ERCC1 showed nearly complete (∼90%) co-localization of the nuclease with sites of (6-4)PP (Fig. 3B). By contrast, the single mutants ERCC1N110A or ERCC1Y145A showed less efficient co-localization with UV lesions. ERCC1N110A was detected at 23%, and ERCC1Y145A was detected at only 9% of the sites of UV damage (Fig. 3C). ERCC1N110A/Y145A was observed at sites of UV damage in less than 3% of the cases (Fig. 3, B and C), even if this protein accumulates in the nucleus. These results further support the conclusion that the reduced NER activity of these ERCC1 mutants is due to their failure to interact with XPA.UV Lesions Persist in Cells Expressing ERCC1N110A/Y145ATo further test the conclusion that the recruitment of ERCC1N110A/Y145A to NER complexes was impaired, we measured the rate of UV lesion repair in ERCC1-deficient UV20 cells and UV20 cells expressing either wild-type ERCC1 or ERCC1N110A/Y145A. Cells were UV-irradiated to generate sites of local DNA damage and fixed at various time points after irradiation, and the amount of damage remaining was assessed by immunodetection of (6-4)PP. Immediately after UV irradiation, 45–55% of the cells contained (6-4)PP, indicating the fraction of cells where a filter pore overlapped with the nucleus (Fig. 4A). At 24 h, 15% of the UV20 cells still stained positively for (6-4)PP (Fig. 4B). This defines the rate of removal of (6-4)PPs from the genome in the absence of NER. (6-4)PPs are likely diluted out during cell division in 24 h (UV20 cells have a doubling time of ∼12 h) or are perhaps eventually removed by other pathways such as homologous recombination. Expression of wild-type ERCC1 in UV20 cells led to a dramatic increase in the rate of (6-4)PP removal, with only 5% of nuclei containing foci 4 h after irradiation and 0% by 8 h after irradiation. By contrast, the slope of the curve indicating removal of (6-4)PP in cells expressing ERCC1N110A/Y145A closely resembled that of untransduced UV20 cells, indicating that NER is severely affected if the XPA-ERCC1 interaction is disrupted.FIGURE 4UV damage persists in UV20 cells expressing ERCC1N110A/Y145A but not wild-type ERCC1. A, untransduced UV20 cells or cells expressing wild-type ERCC1 or ERCC1N110A/Y145A were UV-irradiated as described in the legend for Fig. 3, cultured for 0, 1, 2, 4, 8, or 24 h following UV irradiation, and then fixed and stained for (6-4)PP. B, graphic representation of the percentage of cells with persistent (6-4)PP at various time points. Data represent the average of at least three independent experiments ± S.D. (error bars). 100 cells were counted for each experiment.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Mutations in the XPA-binding Domain of ERCC1 Inhibit NER but Not ICL or DSB RepairHaving established the importance of the XPA-binding domain of ERCC1 for NER, we wished to determine whether this domain is also important for other DNA repair activities of ERCC1-XPF. To test this, UV20 cells expressing wild-type ERCC1, ERCC1N110A, ERCC1Y145A, or ERCC1N110A/Y145A were tested for their sensitivity to UV irradiation, cross-linking agents (mitomycin C and cisplatin), and ionizing radiation by clonogenic survival assays (Fig. 5). As expected, untransduced UV20 cells were hypersensitive to all of these genotoxins relative to parental wild-type AA8 cells. Furthermore, expression of wild-type ERCC1 corrected the sensitivity to all genotoxins. Cells expressing either ERCC1N110A or ERCC1Y145A showed slightly increased sensitivity to UV when compared with wild-type cells and corrected UV20 cells. However, UV20 cells transduced with ERCC1N110A/Y145A were significantly more sensitive to UV than either AA8 or corrected cell lines, although not as sensitive as UV20 cells, demonstrating that mutations in the XPA-interacting domain of ERCC1 affect long term survival following exposure to UV irradiation. Interestingly, all of the ERCC1 mutations studied here supported normal resistance to mitomycin C (Fig. 5B), cisplatin (Fig. 5C), and ionizing radiation (Fig. 5D) at levels comparable with AA8 cells or UV20 cells expressing wild-type ERCC1. These observations provide direct experimental evidence that the ERCC1-XPA interaction is required only for NER and not for ICL and DSB repair activities. The data of the ERCC1N110A/Y145A mutant demonstrate that the functions of ERCC1-XPF in NER can be separated from its functions in ICL and DSB repair by selectively blocking its interaction with XPA.FIGURE 5Mutations in the XPA-binding domain of ERCC1 inhibit NER but not ICL or DSB repair. A–D, clonogenic survival assays to measure the sensitivity of CHO cell lines AA8 (WT) (red diamonds), Ercc1−/− UV20 (blue diamonds), and UV20 expressing ERCC1WT (green diamonds), ERCC1N110A (black diamonds), ERCC1Y145A (purple diamonds), or ERCC1N110A/Y145A (yellow diamonds) to UV-C (A), mytomycin C (MMC) (B), cisplatin (C), or ionizing radiation (D). The data are plotted as the percentage of colonies that grew on the treated plates relative to untreated plates ± S.E. (error bars). cDDP, cis-diamminedichloroplatinum(II).View Large Image Figure ViewerDownload Hi-res image Download (PPT) IntroductionThe preservation of the genetic information contained in DNA is essential for proper cell function and is ensured by multiple DNA repair pathways. Among these, nucleotide excision repair (NER) 2The abbreviations used are: NERnucleotide excision repairICLinterstrand cross-linkXPxeroderma pigmentosumERCCexcision repair cross-complementing(6-4)PP(6-4)photoproductCHOChinese hamster ovaryPBSphosphate-buffered salineDSBdouble strand breakWTwild type. clears the genome of bulky, helix-distorting DNA lesions, such as those formed by UV light, environmental mutagens, and antitumor agents (1Friedberg E.C. Walker G.C. Siede W. Wood R.D. Schultz R.A. Ellenberger T. DNA Repair and Mutagenesis. 2nd Ed. ASM Press, Washington, D. C.2005Crossref Google Scholar, 2Gillet L.C. Schärer O.D. Chem. Rev. 2006; 106: 253-276Crossref PubMed Scopus (488) Google Scholar). Two subpathways of NER exist that differ in their method of damage recognition. In transcription-coupled NER, lesions in the transcribed strand of genes block the progression of RNA polymerase II, triggering NER (3Hanawalt P.C. Spivak G. Nat. Rev. Mol. Cell Biol. 2008; 9: 958-970Crossref PubMed Scopus (761) Google Scholar). In global genome NER, helix-distorting lesions anywhere in the genome are recognized by the XPC-RAD23B heterodimer (4Sugasawa K. Ng J.M. Masutani C. Iwai S. van der Spek P.J. Eker A.P. Hanaoka F. Bootsma D. Hoeijmakers J.H. Mol. Cell. 1998; 2: 223-232Abstract Full Text Full Text PDF PubMed Scopus (741) Google Scholar), in some cases with the help of UV-DDB (5Sugasawa K. Okuda Y. Saijo M. Nishi R. Matsuda N. Chu G. Mori T. Iwai S. Tanaka K. Tanaka K. Hanaoka F. Cell. 2005; 121: 387-400Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar). The subsequent steps of NER are believed to be similar for both subpathways and occur by the sequential assembly of the NER proteins at the site of the lesion (6Volker M. Moné M.J. Karmakar P. van Hoffen A. Schul W. Vermeulen W. Hoeijmakers J.H. van Driel R. van Zeeland A.A. Mullenders L.H. Mol. Cell. 2001; 8: 213-224Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar, 7Riedl T. Hanaoka F. Egly J.M. EMBO J. 2003; 22: 5293-5303Crossref PubMed Scopus (342) Google Scholar, 8Fousteri M. Vermeulen W. van Zeeland A.A. Mullenders L.H. Mol. Cell. 2006; 23: 471-482Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). Recruitment of TFIIH containing two helicase subunits XPB and XPD leads to the separation of the damaged and undamaged DNA strands (9Evans E. Moggs J.G. Hwang J.R. Egly J.M. Wood R.D. EMBO J. 1997; 16: 6559-6573Crossref PubMed Scopus (398) Google Scholar, 10Tapias A. Auriol J. Forget D. Enzlin J.H. Schärer O.D. Coin F. Coulombe B. Egly J.M. J. Biol. Chem. 2004; 279: 19074-19083Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). This enables subsequent NER factors to bind, including XPA, the single-stranded binding protein RPA, and the endonuclease XPG (7Riedl T. Hanaoka F. Egly J.M. EMBO J. 2003; 22: 5293-5303Crossref PubMed Scop" @default.
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• W1992129085 title "The XPA-binding domain of ERCC1 Is Required for Nucleotide Excision Repair but Not Other DNA Repair Pathways" @default.
• W1992129085 cites W1535018042 @default.
• W1992129085 cites W1558439345 @default.
• W1992129085 cites W1765673329 @default.
• W1992129085 cites W1964616851 @default.
• W1992129085 cites W1974691766 @default.
• W1992129085 cites W1975365355 @default.
• W1992129085 cites W1980444814 @default.
• W1992129085 cites W1980819419 @default.
• W1992129085 cites W1981148605 @default.
• W1992129085 cites W1982597025 @default.
• W1992129085 cites W1989208996 @default.
• W1992129085 cites W1989829234 @default.
• W1992129085 cites W1990294201 @default.
• W1992129085 cites W1990849368 @default.
• W1992129085 cites W1992431849 @default.
• W1992129085 cites W1996372461 @default.
• W1992129085 cites W1996516839 @default.
• W1992129085 cites W2004899809 @default.
• W1992129085 cites W2006338892 @default.
• W1992129085 cites W2007771316 @default.
• W1992129085 cites W2016648136 @default.
• W1992129085 cites W2022631460 @default.
• W1992129085 cites W2036680792 @default.
• W1992129085 cites W2040709701 @default.
• W1992129085 cites W2043610941 @default.
• W1992129085 cites W2044000184 @default.
• W1992129085 cites W2046082575 @default.
• W1992129085 cites W2059186992 @default.
• W1992129085 cites W2061353549 @default.
• W1992129085 cites W2061545358 @default.
• W1992129085 cites W2069651971 @default.
• W1992129085 cites W2070821144 @default.
• W1992129085 cites W2071909648 @default.
• W1992129085 cites W2079185082 @default.
• W1992129085 cites W2080605611 @default.
• W1992129085 cites W2080726979 @default.
• W1992129085 cites W2080883315 @default.
• W1992129085 cites W2082398105 @default.
• W1992129085 cites W2083231946 @default.
• W1992129085 cites W2083508435 @default.
• W1992129085 cites W2087954689 @default.
• W1992129085 cites W2091370689 @default.
• W1992129085 cites W2102265131 @default.
• W1992129085 cites W2106515413 @default.
• W1992129085 cites W2106848851 @default.
• W1992129085 cites W2109113345 @default.
• W1992129085 cites W2109468970 @default.
• W1992129085 cites W2112162434 @default.
• W1992129085 cites W2118619700 @default.
• W1992129085 cites W2119882170 @default.
• W1992129085 cites W2120453274 @default.
• W1992129085 cites W2121909403 @default.
• W1992129085 cites W2125625919 @default.
• W1992129085 cites W2128552081 @default.
• W1992129085 cites W2130172167 @default.
• W1992129085 cites W2131348261 @default.
• W1992129085 cites W2141601266 @default.
• W1992129085 cites W2142341469 @default.
• W1992129085 cites W2149940457 @default.
• W1992129085 cites W2150072036 @default.
• W1992129085 cites W2155447972 @default.
• W1992129085 cites W2157004858 @default.
• W1992129085 cites W2157811245 @default.
• W1992129085 cites W2158437043 @default.
• W1992129085 cites W2166424758 @default.
• W1992129085 cites W2170030890 @default.
• W1992129085 cites W2170454539 @default.
• W1992129085 cites W2290020609 @default.
• W1992129085 cites W4249296576 @default.
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