RINT-1, a novel Rad50-interacting protein, participates in radiation-induced G(2)/M checkpoint control.

Rad50, an structural maintenance of chromosomes (SMC) protein family member, participates in a variety of cellular processes, including DNA double-strand break repair, cell cycle checkpoint activation, telomere maintenance, and meiosis. Disruption of Rad50 in mice leads to lethality during early embryogenesis, indicating its essential function in normal proliferating cells. In addition to its ability to form a complex with the DNA double-strand break repair proteins Mre11 and NBS1, Rad50 may interact with other cellular proteins to execute its full range of biological activities. A novel 87-kDa protein named RINT-1 was identified using the C-terminal region of human Rad50 as the bait in a yeast two-hybrid screen. Human RINT-1 shares sequence homology with a novel protein identified in Drosophila melanogaster, including a coiled-coil domain within its N-terminal 150 amino acids, a conserved central domain of about 350 amino acids, and a C-terminal region of 90 amino acids exhibiting 35--38% identity. The conserved central and C-terminal regions of RINT-1 are required for its interaction with Rad50. While Rad50 and RINT-1 are both expressed throughout the cell cycle, RINT-1 specifically binds to Rad50 only during late S and G(2)/M phases, suggesting that RINT-1 may be involved in cell cycle regulation. Consistent with this possibility, MCF-7 cells expressing an N-terminally truncated RINT-1 protein displayed a defective radiation-induced G(2)/M checkpoint. These results suggest that RINT-1 may play a role in the regulation of cell cycle control after DNA damage.

The RAD50 gene was first identified in Saccharomyces cerevisiae based on its role in DNA repair (1). It is a member of the RAD52 epistasis group, members of which function in recombinational DNA repair. Yeast strains carrying rad50 mutations exhibit hypersensitivity to ␥-irradiation and radiomimetic chemicals such as methyl methanesulfonate and only slight sensitivity to UV irradiation (1). In addition, rad50 mutations also lead to telomere shortening and meiotic failure (1). The 153-kDa Rad50 protein contains both an N-terminal Walker A and an C-terminal Walker B NTP binding domains, linked by two extensive coiled-coil regions that are characterized by leucine heptad repeats (2). This sequence architecture is similar to that found in other members of the structural maintenance of chromosomes (SMC) 1 protein family (3,4). The first 35 heptad repeats span amino acids 177-421 of the Rad50 sequence, whereas the second 37 heptad repeats span amino acids 743-995. These heptad repeat sequences exhibit length and significant sequence similarity to the S2 domain of rabbit myosin (2). Mutations within the Rad50 Walker A domain that alter conserved amino acids resides within the NTP binding site confer a null phenotype, and several mutations near the nucleotide binding sites cause defects in meiosis (5).
The RAD50 gene product is highly conserved among species. Human and yeast Rad50 share more than 50% sequence identity within their corresponding N-and C-terminal regions as well as a similar coiled-coil structure in between. A null mutation of the mouse Rad50 gene is associated with an embryonic lethal phenotype, and cells derived from early embryos are hypersensitive to ionizing radiation, suggesting that Rad50 is essential for normal cell proliferation and DNA double-strand break repair (6). Yeast Rad50 forms a complex with Mre11 and Xrs2 (7). Similarly, human Rad50 forms a complex with hMre11 and NBS1, the potentially functional homologue of yeast Xrs2 (8,9). It is believed that the Mre11-Rad50-NBS1 protein complex plays a central role in the cellular response to DNA damage. The NBS1 gene is mutated in Nijmegen Breakage syndrome (9,10), which is phenotypically characterized by radiosensitivity and increased chromosomal instability upon ionizing radiation. Mre11 possesses DNA end-holding as well as endonucleolytic and exonucleolytic activities (11)(12)(13)(14)(15) that may be utilized during the initial steps of DNA double-strand break repair. Mouse embryonic stem cells deficient in Mre11 are not viable (16), suggesting that Mre11, like Rad50, is essential for normal proliferating cells and DNA double-strand break repair. Thus, while it is clear that these three proteins function as a triplex in DNA double-strand break repair, it is also very likely that these proteins function in association with other partners to have different biological functions.
To explore other potential functions of Rad50, we have identified a novel Rad50-interacting protein, named RINT-1. These two proteins specifically interact during S and M phases of the cell cycle. Expression in human breast cancer MCF7 cells of an N-terminally truncated RINT-1 protein that is capable of binding to Rad50 leads to failure of G 2 /M, but not G 1 /S, checkpoint control. These results suggest that RINT-1 may play a role in cell cycle checkpoint control.

EXPERIMENTAL PROCEDURES
Cell Lines, Culture Conditions, and Synchronization-Cell lines including T24 and 5637 (human bladder carcinoma cell lines); Soas-2 (a human osteogenic sarcoma cell line); HeLa (a human cervical carcinoma line); MB231, ZR75, and T47D (human breast cancer lines); SW837 (a rectal carcinoma line); and GM09607A and GM09637G (cell lines with mutated and wild-type ataxia telangiectasia gene (ATM), respectively) (Coriell Cell Repositories) were cultured in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum. Molt-4, a lymphoblastic leukemia cell line, and LEM, a human B cell lymphoma line, were cultured in RPMI 1640 medium plus 10% fetal bovine serum and 5% CO 2 . MCF10A, a breast epithelial cell line, was cultured in Dulbecco's modified Eagle's medium/Ham's F-12 with 20 ng/ml epidermal growth factor, 100 ng/ml cholera toxin, 0.01 mg/ml insulin, 500 ng/ml hydrocortisone, and 5% horse serum. Synchronization of T24 cells by density arrest was described previously (17). Cells were collected at various time points, as G12 (12 h following release from the arrest), G18, G24, and G33. To obtain cells in M phase, nocodazole (0.4 g/ml) was added to the culture medium for 8 h prior to harvest as described (17).
Yeast Two-hybrid Screen and Binding Assays-A pAS-R50PB plasmid was constructed by inserting a portion of the human Rad50 cDNA encoding the region containing second leucine heptad repeats and Walker B NTP binding domain (amino acids 753-1312) into the expression vector pAS1, which contains the GAL4 DNA-binding domain (18,19). pAS-R50PB was used as the bait in a screen of a human B lymphocytes cDNA library as described previously (20). The cDNA clones identified in the screen were sequenced by the dideoxynucleotide termination method (21). For binding assays, three Rad50 deletion constructs were fused with the GAL4 DNA-binding domain in frame, creating pAS-R50PB (amino acids 753-1312), pAS-R50PB0.6 (amino acids 753-953), and pAS-R50PB1.2 (amino acids 953-1312). Various RINT-1 deletion mutants (RINT-H, amino acids 1-227; RINT-RV, amino acids 1-466; RINT-P, amino acids 1-706; RINT-PCR3, amino acids 415-792; and RINT-SX, amino acids 257-792) were fused with GAL4 transactivation domain on the pSE1107 vector. The plasmids containing the bait and the prey were cotransformed into Y153 yeast strain, assayed for ␤-galactosidase activities by the colony lift method, and quantified with the chlorophenol red-␤-D-galactopyranoside assay as described (20).
Plasmid Construction and Expression of Fusion Proteins in Escherichia coli-Plasmids for the bacterial expression of several glutathione S-transferase (GST) fusion proteins were constructed following standard protocols (22). GST-PIN and GST-PIC contain translational fusions of GST with amino acids 1-256 and 501-792 of RINT-1, respectively. For Rad50, GST-PB (amino acids 753-1312 of Rad50), GST-PB0.6 (amino acids 753-953), and GST-PB1.2 (amino acids 953-1312) were constructed. Expression of the fusion proteins was induced by the addition of isopropyl-␤-D-thiogalactopyranoside to a final concentration of 0.1 mM in an exponentially growing culture of bacteria at 30°C for 3 h. Bacteria were collected and lysed as described. Fusion proteins were purified by affinity chromatography using glutathione-coupled agarose beads (23).
In Vitro Binding Assays-For in vitro transcription and translation of RINT-1 protein, the cDNA that begins with the first ATG start codon ( Fig. 1) was subcloned into pBSK and transcribed and translated in vitro using the TNT-coupled reticulocyte lysate system (Promega, Madison, WI). Glutathione-Sepharose beads containing about 5 g of GST or GST fusion proteins were initially preincubated with Tris-buffered saline-bovine serum albumin buffer (25 mM Tris-HCl, pH 8.0, 120 mM NaCl, 10% bovine serum albumin, 1 g/ml of protease inhibitors including leupeptin, antipain, aprotinin, and pepstatin) for 10 min at room temperature. The beads were then incubated with an equal amount of in vitro translated products in lysis buffer (100 mM NaCl, 50 mM Tris-HCl, pH 7.4, 5 mM EDTA, 0.5% Nonidet P-40, and the protease inhibitors listed above) for 1 h at room temperature with rotation. Complexes were washed extensively with the lysis buffer, resolved by SDS-polyacrylamide gel electrophoresis, and detected by fluorography.
Cell Lysate Preparation, Immunoprecipitation, and Immunoblotting-To identify the cellular RINT-1 protein, about 1 ϫ 10 7 Molt-4 cells were metabolically labeled with [ 35 S]methionine for 3 h and lysed in ice-cold Lysis250 buffer (50 mM Tris-HCl, pH 7.4, 250 mM NaCl, 5 mM EDTA, 0.1% Nonidet P-40, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1 g/ml leupeptin, 1 g/ml antipain) (24). Equal amounts of clarified lysates (14,000 ϫ g for 5 min) were incubated with anti-PIN or anti-PIC antibodies at 4°C for 1 h, protein A-Sepharose beads were added, and the mixture was incubated for another 1 h with constant rotation. Finally, the beads were collected and washed with Lysis250 buffer five times, and the immunoprecipitates were separated by 7.5% SDS-polyacrylamide gel electrophoresis and detected by fluorography. For double immunoprecipitations, the immunoprecipitates were boiled in 200 l of dissociation buffer (20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 1% SDS, and 5 mM dithiothreitol), diluted with 1 ml of Lysis250 buffer, and immunoprecipitated once again with the same antibodies as previously described (24).
For coimmunoprecipitation, synchronized T24 cells were lysed and clarified as described above, and the supernatant was diluted with lysis buffer without salt (the final concentration of NaCl was 180 mM) before adding the anti-Rad50 mAb 13B3 (25) or anti-RINT-1 antisera. The immunoprecipitates were separated by SDS-polyacrylamide gel electrophoresis, transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA), and immunoblotted with the indicated antibodies. The immunoblots were then probed with alkaline phosphatase-conjugated secondary antibody and visualized with 5-bromo-4-chloro-3-indolylphosphate toluidinium and nitro blue tetrazolium (Promega, Madison, WI). In some experiments, p84, a nuclear matrix protein (26), served as an internal control.
Isolation of Cell Clones with Expression of the NH 2 -terminal Truncated RINT-1 Protein-To establish cell clones that conditionally express the mutated RINT-1 (named RINT-XS, from amino acid 257 to 792), a UHD10 -3-base plasmid, pUHD10 -3/GFP-RINT-XS, that will express chimeric proteins containing GFP with a nuclear localization signal and a Myc epitope (27) fused to the RINT-XS controlled by a Tet-responsive promoter, was constructed. This plasmid was cotransfected into MCF-7 cells with a second plasmid, pCHTV, which contains a hygromycin resistance gene and a cytomegalovirus-controlled tetracycline repressor-VP16 fusion transcription unit (27). For constitutive expression of the RINT-XS protein, a CHPL-GFP-base plasmid (28), with identical structure of pUHD10 -3/GFP-RINT-XS, except controlled by CMV promoter and with the hygromycin resistance gene as selection marker, was used. Cell clones resistant to hygromycin (100 g/ml) were subsequently isolated and examined for either constitutive or tetracycline-regulated expression of the GFP-RINT-XS proteins. Expression of these fusion proteins in these clones was examined by immunoprecipitation with anti-Myc mAb 9E10 and blotted with an anti-GFP mAb (CLONTECH, Palo Alto, CA). Two constitutively expressing clones, clones 1 and 12, and one conditionally expressing clone, 122, were established and characterized.
Cell Cycle Checkpoint Analysis-Cell cycle checkpoints were determined according to the procedures described (27,29). For the G 1 /S checkpoint assay, cells in logarithmic growth were seeded on coverslips for 24 h in the presence or absence of tetracycline and mock-exposed or treated with 12 grays of ␥-irradiation. After 24 h, cells were incubated with 10 M BrdUrd for 4 h and fixed for BrdUrd staining using a cell proliferation kit (Amersham Pharmacia Biotech). More than 1000 cells were counted for each clone under different conditions, and BrdUrdpositive cells were quantified and expressed as a fraction of the total cells. For the G 2 /M checkpoint, cells were seeded on the six-well dish with a density of 10 5 cells/well in the presence or absence of tetracycline for 24 h. Cells were then irradiated with 4 Gy and fixed with 4% paraformaldehyde at indicated time points. Alternatively, cells were fixed 2 h after treatment with various doses of irradiation. During the fixation process, cells were stained with 4Ј,6-diamidino-2-phenylindole. About 200 -400 cells in a field under a fluorescence microscope were imaged, and the mitotic phase including prophase, metaphase, anaphase, and telophase was counted. More than 6000 cells under different microscopic fields were counted for each time point and expressed as a percentage of mitotic indices compared with the cells without treatment.

RINT-1 Encodes a Novel Human Protein That Shares a Significant Homology with a Putative Drosophila Protein-To
identify proteins that potentially interact with human Rad50, a fragment of Rad50 containing the second of two leucine heptad repeats and the Walker B NTP binding domain was used as bait in a yeast two-hybrid screen of a human B-cell library. The six strongest positive clones identified in this screen were sequenced and found to be identical. Subsequent data base searches revealed that the insert cDNA encodes a novel protein that we named hsRINT-1, which stands for Rad50 interacting protein 1. The complete sequence of the longest cDNA encompasses 2,855 nucleotides and contains a putative translation Rint-1 a Novel Rad50-binding Protein initiation codon, the first methionine, at position 111 with an open reading frame of 792 amino acids (Fig. 1A). With the exception of several leucine heptad repeats in the N terminus of the predicted open reading frame, no other known functional domains have been identified in the predicted 87-kDa protein. However, significant sequence homology was found between RINT-1 and the CG8605 gene product in Drosophila melanogaster (30), which we refer to as dmRINT-1 (Fig. 1B). The predicted sequences of both of these proteins share similar leucine heptad repeats within their corresponding N-terminal region (box A). Two additional regions designated box B, which spans amino acids 220 -565 of hsRINT-1, and box D, which spans amino acids 659 -751 of hsRINT-1, exhibit 35-39% homology to the corresponding regions of dmRINT-1. hsRINT-1 has a unique sequence, box C, that lies between the two conserved regions. The biological function of the dmRINT-1 is unknown at the present time.
A search of the NCBI human genome data base revealed that hsRINT-1 maps to chromosome segment 7q22.1, between marker D7S2545 and D7S2420. Interestingly, deletion of this region is frequently associated with acute myeloid leukemia, myelodysplastic syndrome, breast and ovarian adenocarcinoma, and additional cancer (31).

RINT-1 Interacts Specifically with Rad50 in Yeast Two-hybrid and GST Pull Down
Assays-To test whether RINT-1 specifically binds to Rad50, we sought to identify the binding region(s) of Rad50 responsible for interaction with RINT-1. In addition to the original bait plasmid, pAS-R50PB, two additional plasmids, pAS-R50PB0.6 and pAS-R50PB1.2, that express the second leucine haptad repeat and the Walker B domain of Rad50, respectively, were constructed as yeast GAL4 DNA-binding domain fusions (Fig. 2A). These plasmids were individually cotransformed into S. cerevisiae Y153 with a prey plasmid pSE-RINT-1 that expresses full-length RINT-1. Colorigenic and ␤-galactosidase activity assays indicated that the second leucine heptad repeat, but not the Walker B domain, of Rad50 interacts with RINT-1 ( Fig. 2A). In vitro binding assays using GST fusions of the same regions of Rad50 gave similar results (Fig. 2C). To identify the domain of RINT-1 that interacts with Rad50, a numbers of RINT-1 deletion mutants were engineered as fusions with the GAL4 activation domain, and each mutant was tested for its ability to interact with Rad50, expressed from pAS-Rad50PB, following cotransformation in yeast. Resultant ␤-galactosidase assays revealed that the B, C, and D region of the RINT-1 protein, corresponding to amino acids 257-792, is required for its interaction with Rad50 ( Fig.   FIG. 1. Sequence comparison between hsRINT-1 and dmRINT-1. A, alignment of the predicted amino acids sequence of hsRINT-1 and dmRINT-1. dmRINT-1 is a deduced protein sequence from the CG8605 gene product of D. melanogaster. Circled residues indicate the potential initiation codons that include the first methionine site and two potential non-AUG translational start sites at leucines. The residues demarcating the leucine heptad repeats are underlined. Boxed residues represent identical residues between the two species. The RINT-XS-GFP fusion protein in Fig. 5 includes RINT-1 protein sequences initiating at arginine residue 256 marked with a star. B, schematic diagrams showing homologous regions between human and fly RINT-1. Box A represents the region containing leucine heptad repeats of hsRINT-1 (amino acids 64 -220) and dmRINT-1 (amino acids 13-183). Box C represents the unique region of hsRINT-1 (amino acids 565-659). Protein sequence similarities between box B and box D of human and fruit fly are 35.6 and 38.7%, respectively.
Identification of the RINT-1 Gene Product as an 87-kDa Cellular Protein-To identify cellular proteins encoded by the novel RINT-1 gene, polyclonal antibodies were raised against two RINT-1 protein fragments expressed as GST fusions, GST-PIN (amino acids 1-256) and GST-PIC (amino acids 501-792). Metabolically labeled cellular proteins derived from human acute lymphoblastic leukemia Molt-4 cells were immunoprecipitated with anti-PIN or anti-PIC immune sera or preimmune sera. A protein doublet exhibiting an electophoretic mobility consistent with the predicted 792-amino acid RINT-1 protein was specifically immunoprecipitated with both anti-PIN and anti-PIC antibodies (Fig. 3A, lanes 3 and 8), but not with preimmune antisera. However, preincubation of these two specific antibodies with GST-PIN (lane 5) or GST-PIC (lane 10) antigen, but not GST (lanes 4 and 9), completely abolished the immunoprecipitation of the protein doublet. In addition to the 87-kDa doublet, other cellular proteins were also observed in the immunoprecipitates. Using the rigorous protocol of reimmunoprecipitation, each of the anti-RINT-1 antibodies detected only the 87-kDa protein doublet (lanes 6 and 11).
The RINT-1 protein synthesized by coupled in vitro transcription and translation using a cDNA template that specifies translation initiation from the first methionine in the RINT-1 sequence exhibits a mobility upon SDS-polyacrylamide gel electrophoresis similar to that of the faster migrating cellular RINT-1 protein (Fig. 3A, compare lane 1 with lane 3). To determine whether the slower migrating form of RINT-1 is the product of post-translational modification, immunoprecipitated RINT-1 protein was treated with alkaline phosphatase. No mobility change was observed following phosphatase treatment, suggesting that the slower migrating form may arise from other cellular processes (data not shown). Alternatively, differential initiation of translation using a non-AUG codon could explain the appearance of the slower migrating form of RINT-1 protein. A longer RINT-1 cDNA including sequence upstream of the first ATG codon was in vitro transcribed and translated (Fig. 1A, the amino acid sequence). While the majority of the product thus synthesized in vitro exhibited an electrophoretic mobility corresponding to the faster migrating form, a distinct product exhibiting a mobility corresponding to the slower migrating form of the cellular RINT-1 protein was also detected (Fig. 3B, compare lane 2 with lane 3). These results suggest that the slower migrating form of the RINT-1 doublet corresponds to a polypeptide whose translation begins at an upstream non-AUG codon. Analysis of the 5Ј RINT-1 cDNA sequence for non-AUG start codons (32) revealed the presence of two potential CUG start sites (see the two circled leucines in Fig. 1). Both of these potential codons are flanked by purines at the Ϫ3and ϩ4-positions, indicating that either or both may be used for initiation of translation.
We next examined the expression profile of the RINT-1 protein doublet in a panel of human cell lines. These include a breast epithelial immortalized line MCF10A; three human breast cancer lines, T47D, MB231, and ZR75; two human bladder carcinoma lines, T24 and 5637; a rectal carcinoma line, SW837; and two immortalized fibroblast lines, GM09607A and GM09637G, carrying mutated and wild-type ATM gene, respectively. RINT-1 doublet was detected in all cell lines (Fig.  3C), indicating that RINT-1 is expressed in many different kinds of human cells.
RINT-1 Is Expressed throughout the Cell Cycle but Interacts with Rad50 Only at Late S and G 2 /M Phase-To further explore the interaction of RINT-1 and Rad50 in cells, human bladder carcinoma T24 cells synchronized by density arrest and subsequent release were harvested at different time points. Cell lysates were subjected to immunoprecipitation with anti-RINT-1 or anti-Rad50 antibody, followed by immunoblot analysis using antibodies specific for RINT-1 and Rad50. The specific cell cycle stage(s) from which the lysates were prepared at each time point was determined by probing immunoblots with anti-RB mAb as previously described (17). The amount of total lysate used for each time point was normalized by immunoblotting with an antibody specific for a nuclear matrix protein, p84 (26) (Fig. 4C, bottom panel). While the 87-kDa doublet was detected in immunoprecipitates using anti-RINT-1 antibody during all stages of the cell cycle, an interaction with Rad50 was observed only during late S, G 2 /M, and M phases (Fig. 4A, lanes 4 -6 in the top panel). Reciprocally, an interaction was also detected only after late S phase by immunoprecipitation with anti-Rad50 mAb. Interestingly, both bands of the RINT-1 doublet were coimmunoprecipitated with Rad50, although the slower migrating form of RINT-1 may preferentially bind to Rad50. Nevertheless, the specific interaction between RINT-1 and Rad50 at late S and G 2 /M suggests that RINT-1 may play a role at these time windows during cell cycle progression.
Expression of an N-terminally Truncated RINT-1 Protein Leads to G 2 /M Checkpoint Failure-To test the possibility that RINT-1 functions in DNA damage-induced cell cycle checkpoint control, we established several stable breast cancer MCF7 clonal cell lines that express GFP fusions containing only the Rad50 binding region of RINT-1 (named GFP-RINT-XS). Among these, clone 1 constitutively expressed the GFP-RINT-XS fusion protein, while expression of the fusion protein in clone 122 was conditionally regulated by the presence of tetracycline (Fig. 5). The GFP-RINT-XS protein could be coimmunoprecipitated along with Rad50 (Fig. 5). It is possible that this N-terminally truncated protein may disturb the biological function(s) of the wild-type RINT-1 by binding to Rad50. Hence, we reasoned that this dominant negative mutant of  and 7), anti-PIN (lanes 3-6), or anti-PIC serum (lanes 8 -11). An identical 87-kDa doublet was specifically recognized by both anti-PIN and anti-PIC sera (lanes 3, 4, 6, 8, 9, and 11). The doublet was abolished when immunoprecipitations were performed in the presence of the corresponding antigens (lanes 5 and 10) but not with GST alone (lanes  4 and 9). Only the doublet was detected when the original immunoprecipitates were dissociated first and reimmunoprecipitated by the same antibodies (lanes 6 and 11). The doublet exhibits a mobility similar to that of RINT-1 transcribed and translated in vitro using the cDNA template specifying initiation from the first methionine (lane 1). The antibodies and competitive antigens used in each experiment are indicated above the lanes. IP/IP, double immunoprecipitations with the indicated antibodies; IVT, the in vitro transcribed and translated RINT-1 protein; pre-I, preimmune serum. B, the slower migrating form of RINT-1 is generated by translation starting at non-AUG codons. Lysates of human colon cancer HCT116 cells labeled with [ 35 S]methionine were immunoprecipitated with preimmune serum (lane 1) or anti-PIN antibody (lane 2). Lane 3, in vitro transcribed and translated RINT-1 protein using a full-length cDNA that includes the 5Ј non-AUG codons (leucines) indicated in Fig. 1. Lane 4, in vitro transcribed and translated RINT-1 protein using a cDNA that begins with the first methionine codon indicated in Fig. 1. The inclusion of the 5Ј non-AUG sequences results in a protein doublet that is identical to the cellular proteins. The use of a cDNA template initiating from the first AUG codon in the RINT-1 sequence, however, gives rise to a single protein species, the mobility of which corresponds to the faster migrating species of the cellular RINT-1 doublet. C, the RINT-1 protein is expressed in various cell types. Lysates prepared from the indicated cell lines were directly immunoblotted with anti-PIN antibodies. p84, which is a nuclear matrix protein, was used to normalize total protein amount. RINT-1 might reveal the potential function of RINT-1 in cell cycle progression. To test this possibility, the clonal cell lines expressing the GFP-RINT-XS fusion, as well as a control clonal cell line (5UGN) expressing GFP alone, were examined for the integrity of their respective radiation-induced G 1 /S and G 2 /M checkpoints. Clone 122, the control clone 5UGN, and the parental MCF 7 cells all exhibited reduced BrdUrd-positive cells after treatment with 12 grays of ␥-irradiation, independent of the GFP fusion protein expression (Fig. 6A). These results indicate that expression of the truncated RINT-1 protein did not interfere with the radiation-induced G 1 /S checkpoint. To examine whether these cells exhibit differences in G 2 /M checkpoint control, the mitotic index of each cell culture at various time points after ␥-irradiation was scored (Fig. 6B). In cells expressing GFP alone (5UGN, ϪTet), a sharp reduction in mitotic index was observed, which was similar to that observed in cells without induced expression of GFP alone or GFP-RINT-XS (5UGN or clone 122, ϩTet). In contrast, cells expressing the GFP-RINT-XS fusion protein (clone 122, ϪTet) showed a comparatively delayed and significantly increased mitotic index (Fig. 6B). In a parallel experiment, mitotic cells were scored 2 h following treatment with various doses of ␥-irradiation (Fig. 6C). Clone 122 cells induced to express GFP-RINT-XS exhibited a higher percentage of mitotic cells than cells without induced expression of the fusion protein or GFP alone. Similar results were also obtained using cells constitutively expressing GFP-RINT-XS (data not shown). Collectively, these results suggest that expression of an N-terminally truncated RINT-1 protein retaining its Rad50 binding region leads to an immediate delay of G2/M checkpoint following DNA dam-age. It is thus very likely that the RINT-1 and Rad50 complex may play an important role in control of the G 2 /M checkpoint in response to DNA damage.
In addition to their function in DNA double-strand break repair, recent data have suggested that Rad50, together with Mre11 and NBS1, may also play a role in cell cycle checkpoint control (reviewed in Ref. 33). In yeast, an unrepairable doublestrand break in ku70 mutant cells causes a permanent G 2 /M cell cycle arrest, while mutations in MRE11 suppress the inability of the cells to overcome double strand break-induced cell cycle arrest (34). In NBS cells, a defect in the S phase checkpoint leading to radioresistant DNA synthesis and failure to activate checkpoints at the G 1 /S and G 2 /M boundaries in response to ionizing radiation has been observed (reviewed in Ref. 35). Furthermore, NBS cells exposed to ionizing radiation exhibit a prolonged accumulation of cells in G 2 phase (36). Interestingly, BRCA1, a tumor suppressor linked to familial breast and ovarian cancer (37), was found to associate with Rad50 (25,38). In addition to its participation in transcriptional regulation, cell growth and differentiation, and DNA repair processes, BRCA1 has also been implicated in cell cycle checkpoint control, since deletions of exon 11 of BRCA1 display a defective G 2 /M checkpoint after ionizing radiation and methyl methanesulfonate treatments (39). However, direct evidence to show Rad50 in G 2 /M checkpoint control remains lacking. It is possible that RINT-1 functions independently or, alternatively, in association with a Rad50-BRCA1 complex to ensure appropriate G 2 /M checkpoint control. Regardless, the identification of this novel Rad50-interacting protein represents an important step toward a more complete description of the activities through which proper cell cycle checkpoint control is achieved.