Title: Nibrin Forkhead-associated Domain and Breast Cancer C-terminal Domain Are Both Required for Nuclear Focus Formation and Phosphorylation
Abstract: The Mre11·Rad50·nibrin protein complex plays an essential role in the mammalian cellular response to DNA double-strand breaks. The disorder Nijmegen breakage syndrome (NBS) results from mutations in the NBS1 gene that encodes nibrin, and NBS cells are radiosensitive and defective in S-phase checkpoint activation following irradiation. In response to radiation, nibrin is phosphorylated by Atm, and the Mre11·Rad50·nibrin complex relocalizes to form punctate nuclear foci. The N terminus of nibrin contains a forkhead-associated (FHA) domain and a breast cancer C-terminal (BRCT) domain, the functions of which are unclear. To determine the role of the FHA and BRCT domains in nibrin function, we have performed site-directed mutagenesis of conserved residues in these motifs. Mutations in the nibrin FHA and BRCT domains did not affect interaction with Mre11·Rad50 or nuclear localization of the complex. However, mutation of conserved residues in either domain disrupted nuclear focus formation and blocked nibrin phosphorylation after irradiation, suggesting that these events may be functionally interdependent. Despite an effect on nibrin phosphorylation, expression of the FHA or BRCT mutants in NBS cells restored the downstream phosphorylation of Chk2 and Smc1, necessary for S-phase checkpoint activation. None of the mutations revealed separate functions for the FHA or BRCT domains, suggesting they do not function independently. The Mre11·Rad50·nibrin protein complex plays an essential role in the mammalian cellular response to DNA double-strand breaks. The disorder Nijmegen breakage syndrome (NBS) results from mutations in the NBS1 gene that encodes nibrin, and NBS cells are radiosensitive and defective in S-phase checkpoint activation following irradiation. In response to radiation, nibrin is phosphorylated by Atm, and the Mre11·Rad50·nibrin complex relocalizes to form punctate nuclear foci. The N terminus of nibrin contains a forkhead-associated (FHA) domain and a breast cancer C-terminal (BRCT) domain, the functions of which are unclear. To determine the role of the FHA and BRCT domains in nibrin function, we have performed site-directed mutagenesis of conserved residues in these motifs. Mutations in the nibrin FHA and BRCT domains did not affect interaction with Mre11·Rad50 or nuclear localization of the complex. However, mutation of conserved residues in either domain disrupted nuclear focus formation and blocked nibrin phosphorylation after irradiation, suggesting that these events may be functionally interdependent. Despite an effect on nibrin phosphorylation, expression of the FHA or BRCT mutants in NBS cells restored the downstream phosphorylation of Chk2 and Smc1, necessary for S-phase checkpoint activation. None of the mutations revealed separate functions for the FHA or BRCT domains, suggesting they do not function independently. Nijmegen breakage syndrome (NBS) 1The abbreviations used are: NBS, Nijmegen breakage syndrome; A-T, ataxia-telangiectasia; FHA, forkhead-associated domain; BRCT, breast cancer C-terminal domain; DMEM, Dulbecco's modified Eagle's medium; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; Bicine, N,N-bis(2-hydroxyethyl)glycine; bis-Tris, 2-[bis(2-hydroxyethyl)-amino]-2-(hydroxymethyl)propane-1,3-diol; LDS, lithium dodecyl sulfate.1The abbreviations used are: NBS, Nijmegen breakage syndrome; A-T, ataxia-telangiectasia; FHA, forkhead-associated domain; BRCT, breast cancer C-terminal domain; DMEM, Dulbecco's modified Eagle's medium; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; Bicine, N,N-bis(2-hydroxyethyl)glycine; bis-Tris, 2-[bis(2-hydroxyethyl)-amino]-2-(hydroxymethyl)propane-1,3-diol; LDS, lithium dodecyl sulfate. is a rare autosomal recessive disorder characterized by growth retardation, microcephaly, immunodeficiency, and an increased incidence of lymphoid cancers (1The International Nijmegen Breakage Syndrome Study Group Arch. Dis. Child. 2000; 82: 400-406Crossref PubMed Scopus (259) Google Scholar). Cells from NBS patients are sensitive to ionizing radiation, or other agents that induce DNA double-strand breaks, and display chromosomal instability, frequently involving the immune receptor loci on chromosomes 7 and 14 (1The International Nijmegen Breakage Syndrome Study Group Arch. Dis. Child. 2000; 82: 400-406Crossref PubMed Scopus (259) Google Scholar). In addition, NBS cells have defects in S-phase cell cycle checkpoint control following exposure to ionizing radiation (2Lim D.-S. Kim S.-T. Xu B. Maser R.S. Lin J. Petrini J.H.J. Kastan M.B. Nature. 2000; 404: 613-617Crossref PubMed Scopus (670) Google Scholar). NBS is caused by mutations in the NBS1 gene on chromosome 8q21 that encodes a 754-amino acid protein, nibrin (3Carney J.P. Maser R.S. Olivares H. Davis E.M. Le Beau M. Yates J.R. Hays L. Morgan W.F. Petrini J.H.J. Cell. 1998; 93: 477-486Abstract Full Text Full Text PDF PubMed Scopus (1009) Google Scholar, 4Matsuura S. Tauchi H. Nakamura A. Kondo N. Sakamoto S. Endo S. Smeets D. Solder B. Belohradsky B.H. Der Kaloustian V.M. Oshimura M. Isomura M. Nakamura Y. Komatsu K. Nat. Genet. 1998; 19: 179-181Crossref PubMed Scopus (264) Google Scholar, 5Varon R. Vissinga C. Platzer M. Cerosaletti K.M. Chrzanowska K.H. Saar K. Beckmann G. Seemanova E. Cooper P.R. Nowak N.J. Stumm M. Weemaes C.M.R. Gatti R.M. Wilson R.K. Digweed M. Rosenthal A. Sperling K. Concannon P. Reis A. Cell. 1998; 93: 467-476Abstract Full Text Full Text PDF PubMed Scopus (858) Google Scholar). Patients with NBS have truncating mutations in the NBS1 gene that result in premature termination of nibrin, and their cells fail to make detectable full-length protein (3Carney J.P. Maser R.S. Olivares H. Davis E.M. Le Beau M. Yates J.R. Hays L. Morgan W.F. Petrini J.H.J. Cell. 1998; 93: 477-486Abstract Full Text Full Text PDF PubMed Scopus (1009) Google Scholar, 5Varon R. Vissinga C. Platzer M. Cerosaletti K.M. Chrzanowska K.H. Saar K. Beckmann G. Seemanova E. Cooper P.R. Nowak N.J. Stumm M. Weemaes C.M.R. Gatti R.M. Wilson R.K. Digweed M. Rosenthal A. Sperling K. Concannon P. Reis A. Cell. 1998; 93: 467-476Abstract Full Text Full Text PDF PubMed Scopus (858) Google Scholar). The phenotype of NBS cells suggests a defect in the detection, signaling, or repair of DNA double-strand breaks. Consistent with this possibility, nibrin is found in a complex with Mre11 and Rad50 in vivo, two proteins with established DNA repair functions (3Carney J.P. Maser R.S. Olivares H. Davis E.M. Le Beau M. Yates J.R. Hays L. Morgan W.F. Petrini J.H.J. Cell. 1998; 93: 477-486Abstract Full Text Full Text PDF PubMed Scopus (1009) Google Scholar, 6Dolganov G.M. Maser R.S. Novikov A. Tosto L. Chong S. Bressan D.A. Petrini J.H.J. Mol. Cell. Biol. 1996; 16: 4832-4841Crossref PubMed Scopus (188) Google Scholar). Nibrin interacts directly with Mre11 via sequences in the C terminus of nibrin, and this interaction is required for the nuclear localization of the Mre11·Rad50·nibrin complex (7Desai-Mehta A. Cerosaletti K.M. Concannon P. Mol. Cell. Biol. 2001; 21: 2184-2191Crossref PubMed Scopus (156) Google Scholar). Within the nucleus the Mre11·Rad50·nibrin complex is distributed unevenly, aggregating in nuclear foci that some reports divide into distinct classes (8Maser R.S. Monsen K.J. Nelms B.E. Petrini J.H.J. Mol. Cell. Biol. 1997; 17: 6087-6096Crossref PubMed Scopus (422) Google Scholar, 9Mirzoeva O.K. Petrini J.H.J. Mol. Cell. Biol. 2001; 21: 281-288Crossref PubMed Scopus (333) Google Scholar). Type I foci are observed in untreated cells where they co-localize with PML bodies (9Mirzoeva O.K. Petrini J.H.J. Mol. Cell. Biol. 2001; 21: 281-288Crossref PubMed Scopus (333) Google Scholar, 10Lombard D.B. Guarente L. Cancer Res. 2000; 60: 2331-2334PubMed Google Scholar). Type II and type III foci are induced by agents such as ionizing radiation, which create DNA double-strand breaks, and arise at putative sites of DNA repair (8Maser R.S. Monsen K.J. Nelms B.E. Petrini J.H.J. Mol. Cell. Biol. 1997; 17: 6087-6096Crossref PubMed Scopus (422) Google Scholar, 9Mirzoeva O.K. Petrini J.H.J. Mol. Cell. Biol. 2001; 21: 281-288Crossref PubMed Scopus (333) Google Scholar, 11Nelms B.E. Maser R.S. MacKay J.F. Lagally M.G. Petrini J.H.J. Science. 1998; 280: 590-592Crossref PubMed Scopus (417) Google Scholar). Other cellular proteins involved in DNA damage responses also accumulate at these sites with varying timing and kinetics (12Paull T.T. Rogakou E.P. Yamazaki V. Kirchgessner C.U. Gellert M. Bonner W.M. Curr. Biol. 2000; 10: 886-895Abstract Full Text Full Text PDF PubMed Scopus (1661) Google Scholar, 13Wang Y. Cortez D. Yazdi P. Neff N. Elledge S.J. Qin J. Genes Dev. 2000; 14: 927-939Crossref PubMed Scopus (94) Google Scholar). In addition to nuclear relocalization, nibrin is phosphorylated upon exposure of cells to ionizing radiation. Nibrin phosphorylation is carried out by the Atm protein kinase, mutated in the radiation sensitivity disorder ataxia-telangiectasia (AT), and occurs primarily on two residues, Ser-278 and Ser-343, although other nibrin residues may be targets as well (2Lim D.-S. Kim S.-T. Xu B. Maser R.S. Lin J. Petrini J.H.J. Kastan M.B. Nature. 2000; 404: 613-617Crossref PubMed Scopus (670) Google Scholar, 14Gatei M. Young D. Cerosaletti K.M. Desai-Mehta A. Spring K. Kozlov S. Lavin M.F. Gatti R.A. Concannon P. Khanna K.K. Nat. Genet. 2000; 25: 115-119Crossref PubMed Scopus (407) Google Scholar, 15Wu X. Ranganathan V. Welsman D.S. Heine W.F. Ciccone D.N. O'Neill T.B. Crick K.E. Pierce K.A. Lane W.S. Rathbun G. Livingston D.M. Weaver D.T. Nature. 2000; 405: 477-482Crossref PubMed Scopus (371) Google Scholar, 16Zhao S. Weng Y.-C. Yuan S.-S.F. Lin Y.-T. Hsu H.-C. Lin S.-C.J. Gerbino E. Song M.-H. Zdzienicka M.Z. Gatti R.A. Shay J.W. Ziv Y. Shiloh Y. Lee E.-Y.H.P. Nature. 2000; 405: 473-477Crossref PubMed Scopus (433) Google Scholar). The function of nibrin phosphorylation is unknown but has been reported to be required for radiation-induced nuclear focus formation by the Mre11·Rad50·nibrin complex (16Zhao S. Weng Y.-C. Yuan S.-S.F. Lin Y.-T. Hsu H.-C. Lin S.-C.J. Gerbino E. Song M.-H. Zdzienicka M.Z. Gatti R.A. Shay J.W. Ziv Y. Shiloh Y. Lee E.-Y.H.P. Nature. 2000; 405: 473-477Crossref PubMed Scopus (433) Google Scholar) and for activation of the S-phase cell cycle checkpoint following radiation (2Lim D.-S. Kim S.-T. Xu B. Maser R.S. Lin J. Petrini J.H.J. Kastan M.B. Nature. 2000; 404: 613-617Crossref PubMed Scopus (670) Google Scholar, 16Zhao S. Weng Y.-C. Yuan S.-S.F. Lin Y.-T. Hsu H.-C. Lin S.-C.J. Gerbino E. Song M.-H. Zdzienicka M.Z. Gatti R.A. Shay J.W. Ziv Y. Shiloh Y. Lee E.-Y.H.P. Nature. 2000; 405: 473-477Crossref PubMed Scopus (433) Google Scholar). Recent investigations (17Buscemi G. Savio C. Zannini L. Micciche F. Masnada D. Nakanishi M. Tauchi H. Komatsu K. Mizutani S. Khanna K.K. Chen P. Concannon P. Chessa L. Delia D. Mol. Cell. Biol. 2001; 21: 5214-5222Crossref PubMed Scopus (178) Google Scholar, 18Falck J. Petrini J.H.J. Williams B.R. Lukas J. Bartek J. Nat. Genet. 2002; 30: 290-294Crossref PubMed Scopus (314) Google Scholar, 19Kim S.-T. Xu B. Kastan M.B. Genes Dev. 2002; 16: 560-570Crossref PubMed Scopus (408) Google Scholar, 20Yazdi P.T. Wang Y. Zhao S. Patel N. Lee E.Y.-H.P. Qin J. Genes Dev. 2002; 16: 571-582Crossref PubMed Scopus (406) Google Scholar) have suggested that both arms of the S-phase checkpoint, involving Atm phosphorylation of either Chk2 or Smc1, are dependent on nibrin expression. Despite this knowledge, the function of nibrin is the most poorly understood of the three components of the Mre11-Rad50·nibrin complex. NBS1 mutant alleles identified in NBS patients appear to be hypomorphic, likely obscuring essential functions of nibrin that result in embryonic lethality in mice carrying null mutations of the Nbs1 gene (21Maser R.S. Zinkel R. Petrini J.H.J. Nat. Genet. 2001; 27: 417-421Crossref PubMed Scopus (181) Google Scholar, 22Zhu J. Peterson S. Tessarollo L. Nussenzweig A. Curr. Biol. 2001; 11: 105-109Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). 2K. M. Cerosaletti and P. Concannon, unpublished observations.2K. M. Cerosaletti and P. Concannon, unpublished observations. Unlike Mre11 and Rad50, mammalian nibrin displays only limited sequence similarity with its functional homologue in Saccharomyces cerevisiae, Xrs2 (3Carney J.P. Maser R.S. Olivares H. Davis E.M. Le Beau M. Yates J.R. Hays L. Morgan W.F. Petrini J.H.J. Cell. 1998; 93: 477-486Abstract Full Text Full Text PDF PubMed Scopus (1009) Google Scholar). Two potentially functional domains, a forkhead associated (FHA) domain (residues 24–100) and a breast cancer C-terminal (BRCT) domain (residues 114–182) are readily discernable in the primary sequence of human nibrin (5Varon R. Vissinga C. Platzer M. Cerosaletti K.M. Chrzanowska K.H. Saar K. Beckmann G. Seemanova E. Cooper P.R. Nowak N.J. Stumm M. Weemaes C.M.R. Gatti R.M. Wilson R.K. Digweed M. Rosenthal A. Sperling K. Concannon P. Reis A. Cell. 1998; 93: 467-476Abstract Full Text Full Text PDF PubMed Scopus (858) Google Scholar). These domains are frequently observed in proteins involved in the DNA damage response, although the juxtaposition of an FHA and a BRCT domain appears unique to nibrin (23Huyton T. Bates P.A. Zhang X. Sternberg M.J.E. Freemont P.S. Mutat. Res. 2000; 460: 319-332Crossref PubMed Scopus (131) Google Scholar, 24Durocher D. Jackson S.P. FEBS Lett. 2002; 513: 58-66Crossref PubMed Scopus (311) Google Scholar). FHA domains have been shown to mediate phosphoprotein interactions, such as that between Rad53 and phosphorylated Rad9 or the homodimerization of phosphorylated Chk2, whereas BRCT domains mediate direct protein-protein interactions, such as those involving 53BPI and p53 (25Sun Z. Hsiao J. Fay D.S. Stern D.F. Science. 1998; 281: 272-274Crossref PubMed Scopus (334) Google Scholar, 26Durocher D. Henckel J. Fersht A.R. Jackson S.P. Mol. Cell. 1999; 4: 387-394Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 27Ahn J.-Y. Li X. Davis H.L. Canman C.E. J. Biol. Chem. 2002; 277: 19389-19395Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 28Derbyshire D.J. Basu B.P. Serpell L.C. Joo W.S. Date T. Iwabuchi K. Doherty A.J. EMBO J. 2002; 21: 3863-3872Crossref PubMed Scopus (149) Google Scholar, 29Joo W.S. Jeffrey P.D. Cantor S.B. Finnin M.S. Livingston D.M. Pavletich N.P. Genes Dev. 2002; 16: 583-593Crossref PubMed Scopus (181) Google Scholar). The availability of known ligands for these and other FHA and BRCT domains has facilitated the identification of critical residues for protein interactions mediated by these domains through the use of site-specific mutagenesis and x-ray crystallography. The role of the FHA and BRCT domains in nibrin function has yet to be clearly defined. It is noteworthy that all known mutations in NBS patients truncate nibrin downstream of these motifs, suggesting the FHA and BRCT domains are indispensable for nibrin function (5Varon R. Vissinga C. Platzer M. Cerosaletti K.M. Chrzanowska K.H. Saar K. Beckmann G. Seemanova E. Cooper P.R. Nowak N.J. Stumm M. Weemaes C.M.R. Gatti R.M. Wilson R.K. Digweed M. Rosenthal A. Sperling K. Concannon P. Reis A. Cell. 1998; 93: 467-476Abstract Full Text Full Text PDF PubMed Scopus (858) Google Scholar, 30Resnick I.B. Kondratenko I. Togoev O. Vasserman N. Shagina I. Evgrafov O. Tverskaya S. Cerosaletti K.M. Gatti R.A. Concannon P. J. Pediatr. 2002; 140: 355-361Abstract Full Text PDF PubMed Scopus (60) Google Scholar). We have previously demonstrated that a C-terminal 353-amino acid fragment of nibrin, lacking both the FHA and BRCT domains, failed to restore cell survival following irradiation and nuclear focus formation when expressed in NBS cells (7Desai-Mehta A. Cerosaletti K.M. Concannon P. Mol. Cell. Biol. 2001; 21: 2184-2191Crossref PubMed Scopus (156) Google Scholar). N-terminal truncation of the nibrin FHA domain was also shown to be sufficient to disrupt nuclear focus formation, although the truncation mutant was characterized by protein instability, resulting in a low level of expression and several protein species of different molecular weights (31Tauchi H. Kobayashi J. Morishima K. Matsuura S. Nakamura A. Shiraishi T. Ito E. Masnada D. Delia D. Komatsu K. J. Biol. Chem. 2001; 276: 12-15Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). There is also scant information regarding the proteins that interact with the nibrin FHA and BRCT domains. Maser et al. (32Maser R.S. Mirzoeva O.K. Wells J. Olivares H. Williams B.R. Zinkel R.A. Farnham P.J. Petrini J.H.J. Mol. Cell. Biol. 2001; 21: 6006-6016Crossref PubMed Scopus (182) Google Scholar) demonstrated an interaction between the N terminus of nibrin, including the FHA and BRCT domains, and the E2F1 transcription factor in a yeast two-hybrid analysis, although this interaction constituted only a small portion of endogenous nibrin. Moreover, yeast two-hybrid analysis is unable to detect phosphoprotein interactions such as those mediated by FHA domains. To address the role of protein interactions mediated by the nibrin FHA or BRCT domains in nibrin function, we have performed site-directed mutagenesis of these domains. Targeted mutations were made at residues in the nibrin FHA and BRCT domains that were deemed critical for interaction with their ligands, based on analogy to other previously studied FHA- or BRCT-containing proteins. These mutated constructs were stably introduced into NBS cells by retroviral transduction, and the effects on the DNA damage response were analyzed. We find that disruption of interactions involving either the FHA or the BRCT domain blocks nuclear focus formation by the Mre11·Rad50·nibrin complex in irradiated cells. Surprisingly, these nibrin mutants are not phosphorylated in response to ionizing radiation exposure, but their expression restores the downstream nibrin-dependent phosphorylation of Chk2 and Smc1 necessary for the S-phase checkpoint. Cell Lines—An SV40-transformed fibroblast cell line from an NBS patient, NBS-ILB1, was used as a recipient for all mutant nibrin constructs (33Kraakman-van der Zwet M. Overkamp W.J.I. Friedl A.A. Klein B. Verhaugh G.W.C.T. Jaspers N.G.J. Midro A.T. Eckardt-Schupp F. Lohman P.H.M. Zdzienicka M.Z. Mutat. Res. 1999; 434: 17-27Crossref PubMed Scopus (99) Google Scholar). NBS-ILB1 cells were grown in DMEM (Invitrogen, Carlsbad, CA) supplemented with 15% fetal calf serum (HyClone, Logan, UT), 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). NBS-ILB1 cells stably expressing nibrin transgenes were maintained in the above media with 500 μg/ml G418 (Invitrogen). NBS-ILB1 cells expressing wild type nibrin or the nibrin phosphorylation site mutation S343A have been described previously (14Gatei M. Young D. Cerosaletti K.M. Desai-Mehta A. Spring K. Kozlov S. Lavin M.F. Gatti R.A. Concannon P. Khanna K.K. Nat. Genet. 2000; 25: 115-119Crossref PubMed Scopus (407) Google Scholar, 34Cerosaletti K.M. Desai-Mehta A. Yeo T.C. Kraakman-van der Zwet M. Zdzienicka M.Z. Concannon P. Mutagenesis. 2000; 15: 281-286Crossref PubMed Scopus (36) Google Scholar). Phoenix A retroviral packaging cells were grown in DMEM with 10% heat-inactivated fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin (available at www.stanford.edu/group/nolan). GM637, an SV40-transformed normal human fibroblast cell line, and AT3BI, an SV40 fibroblast cell line from a patient with ataxia-telangiectasia were used as controls and were grown in DMEM, 15% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Site-specific Mutagenesis—Five point mutations, R28A, H45A, D95N, G136E,G137E (labeled as GG136–137EE in the figures), and Y176A, were introduced into the nibrin FHA and BRCT domains using QuikChange site-directed mutagenesis (Stratagene, La Jolla, CA). The corresponding nucleotide changes, 82–83AG → GC, 133–134CA → GC, 283G → A, 407G → A, 410G → A, and 526–527TA → GC, were made in individual NBS1 cDNAs using complementary oligonucleotide primers overlapping the nucleotides to be changed. The following primer sequences were utilized: R28A, 5′-CGTTGAGTACGTTGTTGGAGCGAAAAACTGTGCCATTCTG-3′; H45A, 5′-CAGTCGATCAGCCGAAATGC-TGCTGTGTTAACTGCTAAC-3′; D95N, 5′-CCCGAACTTTGAAGTCGGGGAATGGTATTACTTTTGGAGTG-3′; G136E,G137E, 5′-CAAGCTATATTGCAACTTGAAGAATTTACTGTAAACAATTGGACAG-3′; and Y176A, 5′-CGTCCAATTGTAAAGCCAGAAGCTTTTACTGAATTCCTGCAG-3′. QuikChange PCR was performed on an EcoRI fragment of the NBS1 cDNA that encompassed the FHA and BRCT domains, from –62 of the 5′ untranslated region to nucleotide 536, subcloned in pBluescript (Stratagene). PCR reactions were prepared according to the manufacturer's specifications using 10–20 ng of double-stranded plasmid DNA; 125 ng each of the complementary forward and reverse mutagenic primers; 200 μm each of dATP, dCTP, dGTP, and dTTP; 10 mm KCl; 10 mm (NH4)2SO4;20mm Tris-HCl, pH 8.8; 2 mm MgSO4; 0.1% Triton X-100; 100 μg/ml bovine serum albumin; and 2.5 units of Pfu-Turbo DNA polymerase (Stratagene) in a final volume of 50 μl. PCR was performed at 95 °C for 30 s, 55 °C for 1 min, and 68 °C for 7 min for 16 cycles. The resultant PCR product was incubated with 10 units of DpnI (Roche Applied Science, Indianapolis, IN) at 37 °Cfor1hto digest the parental plasmid template, and 1 μl of the reaction was transformed into XLI-Blu supercompetent bacteria (Stratagene). Resultant recombinant colonies were screened for the appropriate nucleotide mutation by fluorescent sequencing. Individual mutations were introduced into a full-length NBS1 cDNA by subcloning the mutagenized EcoRI fragment into an EcoRI digested full-length NBS1 cDNA in pBluescript (34Cerosaletti K.M. Desai-Mehta A. Yeo T.C. Kraakman-van der Zwet M. Zdzienicka M.Z. Concannon P. Mutagenesis. 2000; 15: 281-286Crossref PubMed Scopus (36) Google Scholar). Retroviral Gene Expression—For retroviral gene expression, a BamHI-NcoI fragment of a mutant NBS1 cDNA was cloned into the HpaI site of the pLXIN retroviral vector (BD Biosciences Clontech, Palo Alto, CA), upstream of the internal ribosome entry site-neomycin cassette. This fragment extended from –62 of the 5′ untranslated region to position 2286 of the NBS1 cDNA, 21 bp 3′ of the stop codon, and just upstream of the polyadenylation signal. Retroviral infection of mutant NBS1 cDNAs was performed as described previously (Ref. 34Cerosaletti K.M. Desai-Mehta A. Yeo T.C. Kraakman-van der Zwet M. Zdzienicka M.Z. Concannon P. Mutagenesis. 2000; 15: 281-286Crossref PubMed Scopus (36) Google Scholar, see also www.stanford.edu/group/nolan). Briefly, 15 μg of retroviral plasmid DNA was introduced into Phoenix A cells using calcium phosphate transfection, and viral supernatants were harvested after 48 h. Following filtration through a 0.45-μm filter, viral supernatants were incubated with NBS-ILB1 target cells for 24 h. Stable bulk cell lines were selected with 1 mg/ml G418 starting 48 h after infection of NBS-ILB1 cells. Western Blot Analysis—Protein expression or modification was analyzed by Western blotting. Total cell lysates were prepared by lysing fibroblast cell lines at a final concentration of 104cells/μl in 1.1× LDS sample buffer (11% glycerol, 155 mm Tris base, 117 mm Tris HCl, 2.2% LDS, 561 μm EDTA, 242 μm Serva Blue G250, 193 μm phenol red) containing 1 mm sodium vanadate and a protease inhibitor mixture (Roche Applied Science). To assess protein phosphorylation following irradiation, cells were exposed to 10 grays (Gy) of ionizing radiation and harvested after 1 h. Alternatively, immunoprecipitates were prepared by lysing 2 × 106 cells in 50 mm sodium phosphate, pH 7.2, 0.5% Triton X-100, 2 mm EDTA, 2 mm EGTA, 25 mm sodium fluoride, 25 mm gylcerophosphate, 2 mm dithiothreitol, 1 mm sodium vanadate, and a protease inhibitor mixture (Roche Applied Science). Lysates were precleared with normal rabbit IgG (Zymed Laboratories Inc., South San Francisco, CA) and GammaBind Plus-Sepharose (Amersham Biosciences, Piscataway, NJ) for 1 h, and then were immunoprecipitated with rabbit polyclonal anti-nibrin antisera (Novus Biologicals, Littleton, CO) and GammaBind Plus-Sepharose overnight. Immunoprecipitates were washed with lysis buffer four times, resuspended in 20 μl of 1.1× LDS lysis buffer, and boiled for 5 min. Prior to gel electrophoresis, 9 parts of cell lysate or immunoprecipitate were mixed with 1 part of 10× NuPAGE sample-reducing agent (Invitrogen) and heated for 10 min at 70 °C. Denaturing gel electrophoresis was performed using either 7% or 3–8% gradient NuPAGE Tris acetate gels (Invitrogen). 105 cell equivalents of total cell lysates or 106 cell equivalents of immunoprecipitates were loaded per lane and electrophoresed at 150 V in Tris acetate SDS running buffer (50 mm Tricine, 50 mm Tris base, 0.1% SDS) with NuPAGE antioxidant (Invitrogen). Following electrophoresis, proteins were transferred to Immobilon P nylon membranes (Millipore Corp., Bedford, MA) in 25 mm Bicine, 25 mm bis-Tris, 1 mm EDTA, 20% methanol with NuPAGE antioxidant. Transfer was accomplished at 30 V for 1 h. Immunoblotting was performed by blocking membranes in 10% nonfat milk in Tris-buffered saline, pH 7.6, with 0.1% Tween 20 (TBST), followed by incubation with primary and secondary antibodies in 5% nonfat milk in TBST. All washing steps were carried out using TBST. For phosphopeptide-specific antibodies, 1% bovine serum albumin in TBST was used for blocking membranes and for antibody dilutions. Primary antibodies used for immunoblotting include rabbit polyclonal antibodies specific for nibrin, Mre11, Rad50, and Chk2 (Novus Biologicals), goat polyclonal anti-Smc1 antisera (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal phosphopeptide antibody specific for nibrin Ser-343 (14Gatei M. Young D. Cerosaletti K.M. Desai-Mehta A. Spring K. Kozlov S. Lavin M.F. Gatti R.A. Concannon P. Khanna K.K. Nat. Genet. 2000; 25: 115-119Crossref PubMed Scopus (407) Google Scholar) and rabbit polyclonal phosphopeptide antibody specific for Smc1 Ser-957, kindly provided by Michael Kastan (19Kim S.-T. Xu B. Kastan M.B. Genes Dev. 2002; 16: 560-570Crossref PubMed Scopus (408) Google Scholar). The primary antibodies were detected with horseradish peroxidase-conjugated goat anti-rabbit IgG (BD Pharmingen, San Diego, CA) or horseradish peroxidase-conjugated donkey anti-goat IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Chemiluminescence was developed using Western Lightning (PerkinElmer Life Sciences, Boston, MA). Immunofluorescence Staining—The detection of nuclear foci following exposure to ionizing radiation was performed as described previously (34Cerosaletti K.M. Desai-Mehta A. Yeo T.C. Kraakman-van der Zwet M. Zdzienicka M.Z. Concannon P. Mutagenesis. 2000; 15: 281-286Crossref PubMed Scopus (36) Google Scholar). Briefly, fibroblast cells lines were grown on glass coverslips (Viromed, Minneapolis, MN) and were irradiated with 12 Gy of ionizing radiation. After 8 h, cells were fixed and permeabilized in 4% paraformaldehyde/0.1% Triton X-100 for 10 min, and coverslips were blocked in phosphate-buffered saline with 10% fetal calf serum overnight. Nibrin and Mre11 expression was detected by co-staining with a polyclonal rabbit antibody to human nibrin (Novus Biologicals) and a monoclonal antibody to human Mre11 (provided by Tony Demaggio, ICOS Corp., Bothell, WA). The primary antibodies were detected using an Alexa 568 goat anti-rabbit IgG conjugate and an Alexa 488 goat anti-mouse IgG conjugate (Molecular Probes, Eugene, OR). Immunofluorescence was analyzed with a Nikon fluorescence microscope and a Bio-Rad confocal imaging system at 488 and 568 nm (Bio-Rad Laboratories, Hercules, CA). To visualize nuclear foci, individual fields were z-planed, 18–20 sections per field, and the sections were stacked to obtain a final image. Mutation of Nibrin FHA and BRCT Domain-conserved Residues—To disrupt the function of the nibrin FHA and BRCT domains, we introduced mutations in amino acid residues that are conserved among FHA and BRCT domains and/or residues that have been demonstrated to be necessary for ligand binding by these domains in other proteins. As shown in Fig. 1A, two conserved amino acids in the nibrin FHA domain, arginine at position 28 and histidine at position 45, were changed to alanine by site-directed mutagenesis. In the x-ray crystal structure of the Rad53 FHA1 domain, these conserved amino acids are located in peptide loops that bind phosphorylated Rad9 (24Durocher D. Jackson S.P. FEBS Lett. 2002; 513: 58-66Crossref PubMed Scopus (311) Google Scholar, 35Durocher D Taylor I.A. Serbassova D. Haire L.F. Westcott S.L. Jackson S.P. Smerdon S.J. Yaffe M.B. Mol. Cell. 2000; 6: 1169-1182Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). The conserved arginine binds the phosphotyrosine residue and the phosphopeptide backbone directly, whereas the conserved histidine residue stabilizes the architecture of the phosphopeptide binding site (35Durocher D Taylor I.A. Serbassova D. Haire L.F. Westcott S.L. Jackson S.P. Smerdon S.J. Yaffe M.B. Mol. Cell. 2000; 6: 1169-1182Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). Mutation of these residues to alanine in Rad53 ablated binding of the FHA1 domain to phosphorylated Rad9 (26Durocher D. Henckel J. Fersht A.R. Jackson S.P. Mol. Cell. 1999; 4: 387-394Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). As a control, we introduced a third mutation in the nibrin FHA domain, D95N. This sequence variant of nibrin was identified among normal individuals and is not associated with any pathologic phenotype (36Cerosaletti K.M. Morrison V.A. Sabath D.E. Willerford D.M. Concannon P. Genes Chromosomes Cancer. 2002; 35: 282-286Crossref PubMed Scopus (45) Google Scholar). In the crystal structure of the Rad53 FHA1 domain, the resid