From the Department of Molecular Medicine and Institute of Biotechnology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78245-3207
Received for publication, September 28, 2000, and in revised form, November 21, 2000
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ABSTRACT |
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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 G2/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 G2/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
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 (11-15) 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 G2/M, but not G1/S, checkpoint
control. These results suggest that RINT-1 may play a role in cell
cycle checkpoint control.
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% CO2. 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 µg/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
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- 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 µg 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 µg/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 × 107 Molt-4 cells were metabolically labeled with
[35S]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 µg/ml leupeptin, 1 µg/ml antipain) (24). Equal amounts of clarified lysates (14,000 × 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 µl 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
NH2-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 µg/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
G1/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
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 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 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 5'
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
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 G2/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,
G2/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 G2/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
G2/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 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 G1/S and G2/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
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 double- strand break in ku70 mutant
cells causes a permanent G2/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 G1/S
and G2/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
G2 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 G2/M checkpoint after ionizing radiation and methyl methanesulfonate treatments (39). However, direct evidence to
show Rad50 in G2/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 G2/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.
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-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).
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-galactosidase activities by the colony lift method, and quantified
with the chlorophenol red-
-D-galactopyranoside assay as
described (20).
-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).
-irradiation. After 24 h, cells were incubated with 10 µM 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
BrdUrd-positive cells were quantified and expressed as a fraction of
the total cells. For the G2/M checkpoint, cells were seeded on the six-well dish with a density of 105 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 4',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.
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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.
-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. 2B). Further deletions of
either RINT-1 B or D region diminished interaction with Rad50 (Fig.
2B).
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Fig. 2.
Interaction between RINT-1 and Rad50.
A, human Rad50 binds RINT-1 in a yeast two-hybrid assay. The
indicated regions of human Rad50 were fused to the DNA-binding domain
of GAL4 in pAS1. RINT-1 was fused to the activation domain of GAL4 in
pSE1107. These plasmids were cotransformed into yeast strain Y153, and
-galactosidase activity was assayed and quantified with the
chlorophenol red-
-D-galactopyranoside. Both of the
leucine heptad repeats of human Rad50 are represented as
solid rectangles. The result indicates that the
second heptad repeat of Rad50 specifically binds to RINT-1.
B, RINT-1 binds human Rad50. The indicated regions of RINT-1
were fused to the activation domain of GAL4 in pSE1107, and these
plasmids were cotransformed with pAS-R50PB containing amino acids
753-1312 of human Rad50 as in A. Note that deletions of
amino acids in the conserved region of RINT-1 (boxes
B and D) abrogated its binding to human Rad50 in
this assay. C, human Rad50 binds RINT-1 by GST pull-down
assay. The expressed and purified human Rad50-GST fusions are shown in
the upper panel. Stars indicate each
corresponding full-length GST fusion protein. In vitro
translated RINT-1 was used to bind to GST fusions, and the results are
shown in the lower panel. The R50PB (amino acids
753-1312) and R50PB0.6 (amino acids 753-953) derivatives bind to
RINT-1 (lanes 2 and 3) but not the GST
or R50PB1.2 (lanes 1 and 4) proteins.
Lane 5 shows 10% of the total input of the
in vitro translated RINT-1.
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Fig. 3.
Characterization of the cellular RINT-1
protein. A, identification of cellular RINT-1 proteins
by two distinct antibodies, anti-PIN and anti-PIC, which recognize
N-terminal and C-terminal regions of RINT-1, respectively. Lysates of
Molt-4 cells labeled with [35S]methionine were
immunoprecipitated with either preimmune serum (lanes
2 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 [35S]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 5' 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 5' 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.
3- and +4-positions,
indicating that either or both may be used for initiation of translation.
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Fig. 4.
Cell cycle-dependent interaction
between RINT-1 and Rad50. Unsynchronized human bladder carcinoma
T24 cells (Un) were arrested at G0 by contact
inhibition (G0), and released by plating at low density for
various time periods (G12 to G33, indicating
hours after release from G0 arrest). M phase cells were
prepared after release for 24 h followed by treatment with
nocodazole. Lysates of T24 cells at each time point were
immunoprecipitated with anti-RINT-1 (A) or anti-Rad50
(B) antibodies. The immunoprecipitates were immunoblotted
with anti-RINT-1 or anti-Rad50 antibodies, respectively (A
and B). C, the lysates were also immunoblotted
with anti-RB mAb to verify the cell cycle status at each time point and
with anti-p84 mAb as a loading control. The RINT-1 doublets were
coimmunoprecipitated with Rad50 during late S (G24),
G2/M (G33), and M phases.
-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 G1/S checkpoint. To examine whether
these cells exhibit differences in G2/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,
Tet), 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, +Tet). In contrast, cells expressing the GFP-RINT-XS fusion protein (clone 122,
Tet) 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 damage. It is thus
very likely that the RINT-1 and Rad50 complex may play an important
role in control of the G2/M checkpoint in response to DNA
damage.
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[in a new window]
Fig. 5.
Expression of an N-terminally truncated
RINT-1 protein derivative that interacts with Rad50. A,
human breast cancer MCF7 cells express the GFP fusions with the
N-terminal truncated RINT-1 protein (RINT-XS). Individual clones were
isolated, and the expression of GFP (CGN#11) or GFP-RINT-XS
(Clone 1) was detected by immunoprecipitation
with anti-Myc-epitope antibody and subsequently blotted with anti-GFP
antibody. Note that the parental MCF cells do not express GFP or the
fusion proteins (lane 1). B, Rad50
binds to RINT-XS in MCF7 clone 1 cells. Cell lysates containing
GFP-RINT-XS fusions were immunoprecipitated with anti-Rad50 antibody
(lane 2) or control anti-GST antibody
(lane 1) and blotted with anti-Rad50
(upper panel) or anti-GFP antibodies
(lower panel) to detect GFP-RINT-XS.
C, clone122 cells express RINT-XS in the absence of
tetracycline. Lysates prepared from clone 122 cells treated with
(lane 1) or without (lane
2) tetracycline were immunoprecipitated by anti-Myc epitope
antibody and blotted with anti-GFP antibody (upper
panel). The parallel lysates were immunoprecipitated with
anti-Rad50 antibody and blotted with either anti-Rad50
(middle panel) or anti-GFP (lower
panel) antibodies. The expressed RINT-XS fusion protein in
clone 122 cells was coimmunoprecipitated by anti-Rad50 antibody.
View larger version (31K):
[in a new window]
Fig. 6.
Cells expressing RINT-XS show defective
G2/M but intact G1/S checkpoints.
A, G1/S checkpoint in -irradiated cells. The
noninduced and induced MCF7 cell clones including 5UGN (GFP alone) and
RINT-XS 122 (GFP fusion protein) were mock-exposed (0 Gy) or treated
with
-irradiation (12 Gy). Cells were labeled 24 h later with
BrdUrd for 4 h and immunostained with anti-BrdUrd antibodies. The
percentage of BrdUrd-positive cells was quantified and presented as a
fraction of the total number of cells. In the
-irradiated cells
expressing GFP or GFP-RINT-XS (
Tet), the relative percentage of
BrdUrd-positive cells decreased as much as the cells without expression
of those proteins (+Tet), indicating an intact
G1/S checkpoint. B and C,
G2/M checkpoint in
-irradiated cells. B,
mitotic indices of cells treated with 4 grays at time points of 1, 2, and 4 h. Over 6000 cells were counted for each time point.
Mock-exposed cells were used as controls. Significant reduction of the
mitotic indices was observed in 5UGN or RINT-XS 122 cells with
tetracycline (+Tet), reflecting an intact G2/M
checkpoint, while RINT-XS 122 cells expressing the GFP-RINT-XS
(
Tet) show marginal reductions of the mitotic indices,
suggesting a defective G2/M checkpoint. C, as
described in B, except cells treated with irradiation of 2, 4, or 8 grays and counted after 2 h.
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ACKNOWLEDGEMENTS |
---|
We thank Yumay Chen, Qing Zhong, Chi-Fen Chen, Shang Li, Lei Zheng, Diane Jones, and Paula Garza for assistance in these experiments and Drs. Z. D. Sharp, Tom Boyer, and Nicholas Ting for critical comments on the manuscript.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants CA81020, CA58183, and EY05758 (to W. H. L.) and Department of Defense Grant BC980052 (to P. L. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The 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 GenBankTM/EMBL Data Bank with accession number(s) AF317622.
These authors contributed equally to this work.
§ To whom correspondence should be addressed. Tel.: 210-567-7351; Fax: 210-567-7377; E-mail: Leew@uthscsa.edu.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M008893200
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ABBREVIATIONS |
---|
The abbreviations used are: SMC, structure maintenance of chromosome; GFP, green fluorescence protein; BrdUrd, bromodeoxyuridine; Tet, tetracycline; GST, glutathione S-transferase; mAb, monoclonal antibody. NTP, nucleoside triphosphate.
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