The Forkhead-associated Domain of NBS1 Is Essential for Nuclear Foci Formation after Irradiation but Not Essential for hRAD50·hMRE11·NBS1 Complex DNA Repair Activity*

NBS1 (p95), the protein responsible forNijmegen breakage syndrome, shows a weak homology to the yeast Xrs2 protein at the N terminus region, known as the forkhead-associated (FHA) domain and the BRCA1 C terminus domain. The protein interacts with hMRE11 to form a complex with a nuclease activity for initiation of both nonhomologous end joining and homologous recombination. Here, we show in vivodirect evidence that NBS1 recruits the hMRE11 nuclease complex into the cell nucleus and leads to the formation of foci by utilizing different functions from several domains. The amino acid sequence at 665–693 on the C terminus of NBS1, where a novel identical sequence with yeast Xrs2 protein was found, is essential for hMRE11 binding. The hMRE11-binding region is necessary for both nuclear localization of the complex and for cellular radiation resistance. On the other hand, the FHA domain regulates nuclear foci formation of the multiprotein complex in response to DNA damage but is not essential for nuclear transportation of the complex and radiation resistance. Because the FHA/BRCA1 C terminus domain is widely conserved in eukaryotic nuclear proteins related to the cell cycle, gene regulation, and DNA repair, the foci formation could be associated with many phenotypes of Nijmegen breakage syndrome other than radiation sensitivity.

NBS1 (p95), the protein responsible for Nijmegen breakage syndrome, shows a weak homology to the yeast Xrs2 protein at the N terminus region, known as the forkhead-associated (FHA) domain and the BRCA1 C terminus domain. The protein interacts with hMRE11 to form a complex with a nuclease activity for initiation of both nonhomologous end joining and homologous recombination. Here, we show in vivo direct evidence that NBS1 recruits the hMRE11 nuclease complex into the cell nucleus and leads to the formation of foci by utilizing different functions from several domains. The amino acid sequence at 665-693 on the C terminus of NBS1, where a novel identical sequence with yeast Xrs2 protein was found, is essential for hMRE11 binding. The hMRE11-binding region is necessary for both nuclear localization of the complex and for cellular radiation resistance. On the other hand, the FHA domain regulates nuclear foci formation of the multiprotein complex in response to DNA damage but is not essential for nuclear transportation of the complex and radiation resistance. Because the FHA/BRCA1 C terminus domain is widely conserved in eukaryotic nuclear proteins related to the cell cycle, gene regulation, and DNA repair, the foci formation could be associated with many phenotypes of Nijmegen breakage syndrome other than radiation sensitivity.
NBS1 is a responsible gene for Nijmegen breakage syndrome (NBS), 1 a variant of ataxia-telangiectasia. Disruption of NBS1 in NBS patients leads to hypersensitivity to ionizing radiation, chromosomal instability, and a predisposition to cancer (1)(2)(3). NBS1 (p95) protein shows a weak (29%) homology to the yeast (Saccharomyces cerevisiae) Xrs2 protein only in the N terminus regions known as forkhead-associated (FHA) domain and BRCA1 C terminus (BRCT) domain (1)(2)(3), which are widely conserved in eukaryotic nuclear proteins related to the cell cycle, gene regulation, or DNA repair (4,5). The protein is known to interact with hMRE11 to form a complex with a nuclease activity for initiation of both nonhomologous end joining and homologous recombination (6,7). However, the function of most of the NBS1 protein is still not understood, because about 70% of the NBS1 protein on the C terminus end does not show any sequence homology to any known proteins including Xrs2 (1)(2)(3). Because the mutations found in NBS patients all occur between codons 220 and 385 of the NBS1 gene (3) and lead to proteins truncated downstream of the FHA/BRCT domain, the C-terminal half of the protein must be associated with the crucial phenotype of NBS, which may depend on nuclear localization of hMRE11⅐hRAD50 (2). Recently, we reported that expression of the full-length NBS1 protein complements multiple NBS phenotype characteristics such as radiation sensitivity, the G 2 checkpoint, and focus formation in nucleus after irradiation (8). These findings enable us to analyze the functional domain of NBS1 using deletion mutants of NBS1 transfected in NBS cells. By this approach, we localized an essential domain at C terminus region of NBS1 for hMRE11 binding. Interestingly, we found that the FHA domain at N terminus of NBS1 is not essential for restoration of radiation resistance of NBS patient cells but is essential for nuclear foci formation after DNA damage by radiation.

EXPERIMENTAL PROCEDURES
Cloning of Chicken Nbs1 cDNA-A partial fragment of the chicken Nbs1 cDNA was obtained from chicken testis poly(A) RNA by RT-PCR using Pyrobest DNA polymerase (TaKaRa) and degenerate primer sets (5Ј-TACGTNGTNGGNMGNAARAA-3Ј and 5Ј-ATGARNGCRCADAT-NGTYTT-3Ј). A fragment representing the FHA/BRCT domains was cloned after two sets of PCR reactions, subcloned into a pBluescript® II KS(Ϫ) vector (Stratagene), and sequenced. The full-length cDNA was then cloned with the 3Ј-and 5Ј-rapid amplification of cDNA ends method.
Plasmid Construction-Construction of the NBS1 expression vector and its transfection into cells were performed as described previously (8). Briefly, NBS1 cDNAs containing C-terminal deletions were amplified by PCR using primers containing BamHI sites and point mutations introducing in-frame termination codons (the forward primer was 5Ј-A-ATATGGATCCTGGACCGATGTGGAAACTGCT-3Ј, and reverse primers were as follows: S744, 5Ј-ATAAGGGGATCCTCAAAAAAGATCAT-CAGCAAGAG-3Ј; S703, 5Ј-TAGATCGGATCCTCAAATGATGTGTGG-AAGTTTTG-3Ј; S670, 5Ј-GCCAGGGATCCTTCAGGAAGTAGAGTTTT-TAATCAC-3Ј; and S590, 5Ј-CCTTGTGGATCCTCAAACATTGACATC-TTCCTC-3Ј) and Pyrobest DNA polymerase (TaKaRa). For construction of FHA or FHA/BRCT deletions, the forward primer was substituted with FE3 (5Ј-TTAATGGGATCCACATGCAGAATGGCTTTTCCCG-3Ј) or BRCT-d (5Ј-TGCTGGATCCTTGTCATGGTATCAGTGAAA-3Ј), and a reverse primer for full-length cDNA (8) was used in the PCR reaction. Amplified cDNA was BamHI-digested and ligated into the BamHI site * This work was supported in part by the Ministry of Education, Science, Sports and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AF230342.
For construction of yeast two-hybrid vectors, full-length NBS1 cDNA or hMRE11 cDNA was ligated into pAS2-1 (CLONTECH) or into the GAL4-activating domain of pACT2 (CLONTECH). Partial deletion mutant plasmids were constructed by PCR using full-length constructs as a template, Pyrobest DNA polymerase (TaKaRa), and oligonucleotides (22-24-mer) designed to make an in-frame deletion. The PCR products were then self-ligated, and the entire DNA sequence was verified.
Cell Culture and Transfection-GM7166VA7 cells from an NBS patient were used as an NBS cell line. HeLa cells were used as a control cell line with normal radiation sensitivity. Cell cultures were maintained in DMEM (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (HyClone). The vectors were transfected into GM7166VA7 cells by electroporation using a GenePulser (Bio-Rad), and stable transformants were selected by incubation in medium containing 200 g/ml hygromycin B (Wako).
Cell Survival Assay-Exponentially growing cells were trypsinized, re-suspended in DMEM, and sealed in a glass tube. The cells were then irradiated with 60 Co ␥ rays at a dose rate of 1.0 Gy/min. Immediately after irradiation, an appropriate number of cells were plated in DMEM supplemented with 10% fetal bovine serum and 10% fetal calf serum. After 14 days of incubation, the cells were fixed with ethanol and stained with a 4% Giemsa solution (Katayama Chemical). Surviving fractions were calculated by comparing the number of colonies in the experimental cells with the number of colonies formed in nonirradiated control cells.
Western Blotting and Immunofluorescent Staining-Whole-cell extracts (from 2 ϫ 10 6 cells) were prepared as described (8), and 40 g of total protein was applied to an 8% SDS polyacrylamide gel. After electrophoresis, proteins were transferred to a blotting membrane using an electroblot apparatus (ATTO), and immunoblots were performed as described previously (8).
For immunofluorescent staining, cells grown on a glass slide were fixed with cold methanol for 20 min, rinsed with cold acetone for several seconds, and then air-dried. The slides were stained as described previously (8). The primary antibodies used were as follows: anti-NBS1 (8), anti-hMRE11 (Novus Biologicals), and anti-hRAD50 (GeneTex). Alexa-488-conjugated anti-rabbit IgG (Molecular Probes) was used for visualization of NBS1 or hMRE11. Biotinylated anti-mouse IgG (Vector) and Alexa-488-conjugated streptavidin (Molecular Probes) were used for hRAD50. The 488-nm excited green fluorescence from the Alexa-488 dye was visualized with a laser scanning microscope (Olympus).
Yeast Two-hybrid Analysis-Full-length or mutated NBS1 cDNA was expressed as a fusion protein to a GAL4-DNA-binding domain (BD) from pAS2-1 (CLONTECH) or to a GAL4-activating domain (AD) from pACT2 (CLONTECH). The full-length or mutated NBS1-BD (or -AD) plasmid was transfected into the yeast strain GC-1945 (CLONTECH), along with a full-length hMRE11-AD (or -BD) plasmid. Interaction between the expressed proteins was detected by growth on a synthetic dropout (ϪLeuϪTrpϪHis) plate and by ␤-galactosidase activity.

RESULTS
To locate the functionally important domain of the NBS1 gene involved in the human NBS phenotype, we tried to determine which sequences were conserved in NBS1 in higher eukaryotes. Cloning and sequencing of the chicken Nbs1 gene can be very useful for locating a conserved domain, because a low homology of the NBS1 amino acid sequence between the human sequence and the chicken sequence has been suggested (2). The amino acid sequence of chicken Nbs1 shows apparent homology with human and mouse NBS1 (62 and 63% identity, respectively) at the N terminus 360 amino acids, which contains both the FHA/BRCT domain and a phosphorylation site on a serine residue at 278 and 343 (Refs. 9 and 10 and data not shown). There is a region with low homology from 360 to 630 (33% identity between chicken and human). Another conserved sequence was observed at the region from 631 to 754 and the C terminus of the protein (Fig. 1). A novel identical sequence with yeast Xrs2 protein was also found in this small region (Fig. 1). This is consistent with the suggestion that the C-terminal half of NBS1 may interact with hMRE11 (2). To confirm this, we generated various C-terminal deletion mutants of NBS1 and tested their ability to bind to hMRE11 using a yeast two-hybrid analysis. Significant interaction between NBS1 and hMRE11 was detected only when codons 665-693 were present in the construct even when N-terminal FHA/BRCT domains were deleted (Fig. 2). This C-terminal region contains a sequence highly conserved at codons 682-693 in the chicken, mouse, human NBS1, and yeast Xrs2 proteins, implying that the sequence might be a critical region for hMRE11 binding.
Because NBS1 is essential for the hMRE11⅐hRAD50⅐NBS1 complex to express nuclease activity or ATP-dependent DNA unwinding activity (11), the hMRE11-binding domain is probably necessary for the processing of damaged DNA. To assay the functions of the binding domain in vivo, we subcloned mutant NBS1 constructs into expression vectors (shown in Fig.  2) and transfected them into the NBS cell line GM7166VA7. All of the stable transformants expressed a significant amount of the mutant NBS1 protein (Fig. 3a). Although the expression of multiple smaller proteins were observed in the N-terminaldeleted mutants (Fig. 3a, FE3 and BRCT-d), the expected mutant NBS1 proteins in the FE3 and BRCT-d clones were still detected in their transformants (Fig. 3a). Because it is known that NBS1 directly binds to hMRE11 and indirectly to hRAD50 through hMRE11 (2), we tested the ability of mutant NBS1 to form the complex. Full-length and S703 mutant protein, containing C-terminal conserved region, were able to form the triple-protein complex, because they coimmunoprecipitated with hRAD50. On the other hand, S590 and S670, in which the C-terminal conserves sequence was deleted, were not able to form the complex (Fig. 3b). The result is consistent with yeast two-hybrid experiment (Fig. 2), which demonstrated the essential C-terminal domain for hMRE11 binding at 665-693. Although the expected mutant protein from FE3 or BRCT-d was invisible in NBS1-blot for hRAD50 precipitates (Fig. 3b), a very weak signal was detected when the increased amount of the precipitate was used for analysis (data not shown). Because the expected FE3 or BRCT-d proteins were accompanied by the degraded small fragments (Fig. 3a), the N terminus mutant proteins could be unstable. The absence of the N terminus region of the protein in the FE3 and BRCT-d clones was confirmed by immunoblotting using antiserum that recognizes the N-terminal end of NBS1 (data not shown). From these results, it appears that the multiple proteins expressed in the FE3 and BRCT-d mutants must contain functional hMRE11-binding domains at the C-terminal NBS1 region.
Because restoration of radiation resistance was observed only when the mutant proteins contained the hMRE11-binding domain at C terminus (Fig. 4), this suggests that the hMRE11binding domain is essential for radiation resistance. This was supported by finding that del 683-693 mutant lacking Xrs2identical sequence could not restore radiation resistance (data not shown). Interestingly, cells transfected with mutant NBS1 lacking the FHA domain alone or lacking both the FHA and BRCT domains (FE3 and BRCT-d) also became radiation resistant to a degree similar to that seen in C-terminal enddeleted mutant (S703 in Fig. 4). These results imply that the FHA/BRCT domain is not essential for restoration of radiation resistance, i.e. for DNA repair.
Subsequently, observations were made to see whether the various truncated NBS1 proteins could restore the focus formation activity of the hRAD50⅐hMRE11⅐NBS1 complex in NBS cells after irradiation, because it has been reported that this  (1-3). Right, the hMRE11 binding activity of various NBS1 constructs by means of yeast two-hybrid analysis. The columns NBS1⅐MRE11 or MRE11⅐NBS1 represent that NBS1 fused to GAL4-DNA-BD (or NBS1 fused to GAL4-AD) was coexpressed with a full-length hMRE11 fused to AD (or BD) in yeast strain GC-1945 (CLONTECH). ␤-Galactosidase activities of each transfectant are indicated by the following symbols: ϩϩ, color change within 1 h; ϩ, color change within 3 h; Ϫ, no color change or weak color change over 12 h; U, undetermined. Note that a weak color change for S670-AD⅐hMRE11-BD (and for del 682-693) was detected after a 24-h incubation. The black box represents the putative domain that is essential for hMRE11 binding.  triple-protein complex forms foci at DNA double-strand breaks in the nucleus after exposure to ionizing radiation (12). An absence of hMRE11 and hRAD50 in the nucleus was observed in NBS cells (GM7166VA7), and foci did not form when cells were irradiated with ␥ rays (Fig. 5, GM7166VA7; see Ref. 2). In contrast, nuclear localization of NBS1, hMRE11, and hRAD50 was detected in NBS cells transfected with full-length NBS1, and foci formation in the nucleus was clearly observed after irradiation (Fig. 5, ϩFull). A mutant of NBS1 containing the hMRE11-binding domain showed nuclear localization of hMRE11 and hRAD50 (Fig. 5, ϩS703, ϩFE3, and ϩBRCT-d), and the lack of the hMRE11-binding domain in the S670, S590, or the del 682-693 clone resulted in the cytoplasmic localization of the proteins. Because the cells in the absence of triple complex in nuclei (S590, S670, and del 682-693) remained radiation-sensitive ( Fig. 4 and data not shown), these results suggest that the nuclear localization of hMRE11 and hRAD50 is necessary to restore the radiation resistance. Surprisingly, the FE3 and BRCT-d cells could not form foci in response to DNA damage (Fig. 5, ϩIR lanes, ϩFE3 and ϩBRCT-d) even though the triple-protein complex was able to localize in the nucleus. To confirm the inability of nuclear foci formation in FE3 mutant NBS1 protein, we transfected this mutant cDNA into HeLa cells. Significant reduction of foci formation after radiation was observed in FE3-expressed HeLa cells, possibly by dominant negative effects (Fig. 6). This FE3-expressing HeLa cell clone showed no alteration of radiation sensitivity (data not shown), supporting the finding that FHA domain is not essential for restoration of radiation resistance. DISCUSSION NBS1 is reported to be essential for nuclear localization of hRAD50⅐hMRE11 complex (2) and to enhance the nuclease activity of the complex (7,11). These observations suggest the function of NBS1 as a key regulator of both localization and activity of the triple-protein complex. We identified the essential region at codons 665-693 of NBS1 for hMRE11 binding. The present results also showed that the FHA/BRCT domain of NBS1 is essential for nuclear foci formation after DNA damage but not for cellular survival after irradiation. This is confirmed by evidence that foci formation was repressed in FE3-transfected HeLa cells. Zhao et al. (10) reported that the alteration of the phosphorylation site at both Ser 273 and Ser 343 residues markedly reduced the foci formation and radiation resistance, suggesting phosphorylation of NBS1 is necessary for both foci formation and DNA repair after irradiation. It is consistent with our result that the expression of a mutant NBS1 protein lacking both phosphorylation site and FHA/BRCT domain in GM7166VA cells was unable to complement not only nuclear foci formation of the complex but also radiation resistance of the cells (data not shown). Therefore, we conclude that FHA/ BRCT domain, possibly sole FHA domain, is essential for the nuclear foci formation of the triple-protein complex together with the presence of both hMRE11-binding domain and the serine residues for phosphorylation.
A number of DNA repair-related proteins are known to form nuclear foci in response to DNA damage, such as RAD51, BRCA1, and BLM, as well as the hRAD50⅐hMRE11⅐NBS1 complex (1,8,12,13), and these might be affected or regulated by phosphorylation signals. In view of this, the failure of the triple-protein complex in the FE3 and BRCT-d clones to form nuclear foci supports the putative functions of the FHA and BRCT domains, namely the FHA domain motif is for proteinprotein interactions that recognize the phosphorylation state of the target protein (14), and the BRCT domain might provide a DNA-binding domain for repair-related proteins (15). The results shown here indicate that FHA/BRCT domain is essential for nuclear foci formation activity following DNA damage, even though they are not directly related to the DNA repair ability itself. This finding is consistent with the fact that most of the DNA double-strand breaks are rejoined within the first hour after irradiation (16), but foci formation persists even 5-8 h after irradiation (8,12). Taken together, it is suggested that the nuclear foci formation is not a strict hallmark of DNA repair.
Because the FHA/BRCT domain is conserved in eukaryotic NBS1 homologue, they might be involved in other crucial phenotypes of NBS, such as in insuring the fidelity of the rejoined DNA.