Mutagenesis Advance Access originally published online on February 8, 2005
Mutagenesis 2005 20(1):57-63; doi:10.1093/mutage/gei011
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Mutagenesis vol. 20 no. 1 © UK Environmental Mutagen Society 2005; all rights reserved.
Nuclear localization of Rad51B is independent of Rad51C and BRCA2
Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, 7000 East Avenue, L-448, Livermore, CA 94550, USA
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Rad51B is one of the five paralogs of human Rad51 and is found in a multiprotein complex with three other Rad51 paralogs, Rad51C, Rad51D and Xrcc2. Participation of Rad51B in this complex depends on its direct interaction with Rad51C. Examination of EGFP–Rad51B fusion protein in HeLa S3 cells and immunofluorescence in several human cell lines reveal the nuclear localization of Rad51B. Mutations in the N-terminal KKLK motif of Rad51B (amino acids 4–7), result in the cytoplasmic localization of Rad51B suggesting that the KKLK sequence is the nuclear localization signal (NLS) for the Rad51B protein. Examination of wild-type EGFP–Rad51B fusion protein in hamster irs3 mutant cells, deficient in Rad51C, showed that Rad51B localizes to the nucleus independently of Rad51C, the only known direct binding partner for Rad51B. Utilization of a BRCA2 mutant cell line, CAPAN-1, showed that Rad51B also localizes to the nucleus independent of BRCA2. Although both Rad51B and BRCA2 are clearly involved in the homologous recombinational repair pathway, Rad51B and BRCA2 do not appear to associate. This study finds that a KKLK motif in the N-terminus of Rad51B serves as an NLS that allows Rad51B to localize to the nucleus independent of Rad51C or BRCA2.
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Introduction |
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Repair of DNA double-strand breaks in eukaryotic cells occurs primarily through two pathways, error prone non-homologous end joining (NHEJ) or high fidelity homologous recombinational repair (HRR) (Thompson and Schild, 2002
; West, 2003). The HRR pathway involves the participation of many proteins including members of the Rad52 epistasis group: Rad51, Rad52, Rad54 and the Rad51 paralog proteins (Thompson and Schild, 2001). Rad51 is central to the catalysis of DNA strand exchange between identical DNA strands in an ATP-dependent manner (Sung and Robberson, 1995). There are five human paralogs of Rad51: Rad51B (Rad51L1), Rad51C (Rad51L2), Rad51D (Rad51L3), Xrcc2 and Xrcc3 (Tebbs et al., 1995; Albala et al., 1997; Rice et al., 1997; Tambini et al., 1997; Cartwright et al., 1998a,b; Dosanjh et al., 1998; Liu et al., 1998; Pittman et al., 1998). Rad51B has been shown to bind directly to Rad51C, and indirectly with Rad51D and Xrcc2 to form a multiprotein complex (BCDX2) that functions in HRR (Masson et al., 2001; Liu et al., 2002; Miller et al., 2002; Wiese et al., 2002). Recently, Rad51C when present in a complex with either Rad51B, Xrcc3 or BDX2 was shown to promote the resolution of Holliday junction-like structures (Liu et al., 2004). Rad51B preferentially binds to synthetic Holliday junction DNA substrates in vitro (Yokoyama et al., 2003), further suggesting that it may have a role in Holliday junction resolution. The BCDX2 complex preferentially binds to synthetic Holliday junctions and to branched DNA structures, supporting that this complex is critical for maintaining genomic stability (Yokoyama et al., 2004). It has also been demonstrated that deficiency of any of the Rad51 paralogs in vertebrate cells results in increased sensitivity to DNA cross-linking agents and ionizing radiations (Cartwright et al., 1998b; Liu et al., 1998; Griffin et al., 2000; Takata et al., 2000, 2001; Godthelp et al., 2002, Drexler; et al., 2004), similar to mutants of Rad51 and BRCA2 [reviewed in Thompson and Schild (2002)].
The BRCA2 breast cancer susceptibility protein, functions in the HRR pathway (Xia et al., 2001) and interacts directly with Rad51 (Chen et al., 1998a). BRCA2 binds to Rad51 through a series of eight similar sequence motifs of 
30 amino acids each, referred to as the BRC repeats (Bork et al., 1996). X-ray crystallography has been used to detail the interaction of Rad51 with the BRC4 repeat of BRCA2 (Pellegrini et al., 2002). It has been suggested that BRCA2 is responsible for loading Rad51 onto single-stranded DNA to create nucleoprotein filaments (Shin et al., 2003). A cancer-causing truncation mutant of the BRCA2 protein that is unable to localize to the nucleus is associated with reduced Rad51 nuclear localization and foci formation following DNA damage (Yuan et al., 1999; Davies et al., 2001), suggesting that Rad51 relies on its interaction with BRCA2 for proper subcellular localization. However, both Rad51 association with chromatin and replication-associated Rad51 foci still occur in CAPAN-1 cells, which carry the BRCA2 truncation (Chen et al., 1998b; Tarsounas et al., 2003).
The nuclear localization of a protein involves the recognition of targeting signals by members of the importin family and subsequent translocation by the nuclear pore complex [reviewed in Jans et al. (2000)]. There are three classes of short modular peptide sequences, each of which is sufficient for nuclear localization. The first of these is a short stretch of basic amino acids; the second contains two short stretches of basic amino acids separated by a spacer of 10–12 amino acid residues (Kalderon et al., 1984; Robbins et al., 1991); and the third contains charged or polar residues interspersed with non-polar residues (Makkerh et al., 1996).
Rad51B has the N-terminal basic residues KKLK which have been suggested to act as a nuclear localization signal (NLS) for this protein (Rice et al., 1997). A KKLK motif is found to independently localize the Ku80 DNA repair protein to the nucleus, demonstrating the functionality of this motif as an NLS (Koike et al., 1999). Recently, it was shown that Rad51C contains an NLS at the extreme C-terminus of the protein (French et al., 2003). Although Rad51D and Xrcc2 are found in nuclear extracts (Braybrooke et al., 2000), it is not yet known whether they are transported independently to the nucleus. Furthermore, previous studies from our laboratory have demonstrated that Rad51B and Rad51C co-immunoprecipitate from HeLa S3 nuclear extracts (Miller et al., 2002). In the present study, hamster cells deficient in Rad51C (French et al., 2002; Jones et al., 1987) showed that Rad51B localizes to the nucleus independently of Rad51C, further suggesting that Rad51B, like Rad51C, contains its own NLS which we have determined to be a KKLK motif at the N-terminus, amino acids 4–7 of Rad51B. In this study, we examine the requirements for Rad51B nuclear localization and show that this localization is independent of both BRCA2 and its binding partner, Rad51C.
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Materials and methods |
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Cell culture
HeLa S3, MCF10A, CAPAN-1 and MCF7 cells were obtained from the American Type Culture Collection (Manassas, VA). HeLa S3 cells were cultivated in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 300 µg/ml L-glutamine and antibiotics. MCF10A cells were cultured in DME:F12 supplemented with 5% heat-inactivated equine serum, 0.01 mg/ml bovine insulin (Sigma, St Louis, MO), 0.5 µg/ml hydrocortisone and 20 ng/ml murine EGF (Becton-Dickinson, Franklin Lakes, NJ), 300 µg/ml L-glutamine and antibiotics. MCF7 cells were grown in Dulbecco's modified Eagle's media
supplemented with 10% heat-inactivated FBS, 300 µg/ml L-glutamine, 0.01 mg/ml insulin and antibiotics. CAPAN-1 cells were grown in RPMI 1640 supplemented with 20% heat-inactivated FBS, 300 µg/ml L-glutamine and antibiotics. V79 hamster cells and the Rad51C-deficient irs3 cells (French et al., 2002) were grown in monolayer culture in -MEM supplemented with 10% heat-inactivated FBS and antibiotics. Cells were maintained in a humidified incubator at 37°C and 5% CO2. All media and supplements were obtained from Invitrogen (Carlsbad, CA) unless otherwise specified.
Primary antibodies
Generation of a polyclonal antibody to Rad51B was previously described (Miller et al., 2002). Additional antibodies used in these studies include a BRCA2 monoclonal Ab-1 (Oncogene Research, San Diego, CA), an actin monoclonal antibody (Oncogene Research), anti-Xrcc2 E17 (Santa Cruz Biotechnology; Santa Cruz, CA), monoclonal antibodies to Rad51C and Rad51D (Novus Biologicals; Littleton, CO), and polyclonal antibodies RAD51 C-20 (Santa Cruz Biotechnology) and RAD51 Ab-1 (Oncogene Research).
Cellular lysates and immunoprecipitation
Cell extracts were prepared from exponentially growing cells in lysis buffer (50 mM Tris, pH 7.5, 50 mM NaCl, 0.5% NP-40 with protease inhibitors), incubated on ice for 30 min, centrifuged at 14 000 g for 5 min to pellet out cellular debris and the supernatants were collected. One milligram of whole cell extract was precleared with 30 µl of recombinant protein G agarose (Invitrogen). Samples were incubated with 25 µg of appropriate antibody or antibody preadsorbed with the appropriate peptide (50 µg). Preimmune rabbit serum was used as a control for the anti-Rad51 immunoprecipitation and a non-related anti-actin monoclonal antibody was used as a control for the anti-BRCA2 monoclonal antibody immunoprecipitation. For each sample, 30 µl of recombinant protein G agarose was added, the samples were incubated for 1 h and then washed three times with 500 µl of cold lysis buffer. Subsequently, 2x SDS–PAGE loading buffer was added and the samples were boiled for 5 min prior to SDS–PAGE.
Western blot analysis
Immunoprecipitated samples or cell lysates were subjected to electrophoresis in 3–8% NuPAGE gels for BRCA2 immunoblots or 12% Tris–Glycine gels (Invitrogen) for Rad51 and Rad51B immunoblots as per the manufacturer's instructions. The resolved polypeptides were transferred to a polyvinylidene difluoride (PVDF) membrane and blocked in 5% milk in phosphate-buffered saline (PBS) for 30 min followed by incubation with the appropriate primary antibody diluted in 5% PBS milk for at least 1 h at room temperature. The Rad51B polyclonal antibody was diluted 1 : 1000, BRCA2 Ab-1 diluted 1:500 and Rad51 C-20 diluted 1:1000. Blots were washed three times with 1x TBS–T [Tris-buffered saline with 0.1% Tween 20 (Bio-Rad, Hercules, CA)] and a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) diluted in 5% PBS milk was applied for 1 h. The membranes were washed three more times in 1x TBS–T and visualized by enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech, Piscataway, NJ).
For the subcellular localization analysis by western blotting, nuclear and cytoplasmic extracts were prepared using the NE-PER kit (Pierce Biotechnology, Rockford, IL) and separated by PAGE on a 12% gel after quantitation by the Bradford method and normalization for loading. Proteins were transferred onto the ECL membrane (Amersham Pharmacia Biotech) and blocked in 5% milk in TBS–T overnight at 4°C. Blots were probed with primary antibody (Rad51C: 1:1000; Rad51D: 1:2000; Xrcc2: 1:500) for at least 2 h and washed with TBS–T prior to incubation with secondary antibody. Secondary antibody incubation and detection were conducted using the ECL detection kit (Amersham Pharmacia Biotech).
Immunofluorescence
For immunocytochemical analysis, cells were grown on coverslips for 24 h, fixed with ice-cold acetone for 2 min and allowed to dry. The coverslips were then incubated overnight at 4°C with the appropriate primary antibody diluted 1:100 in 5% PBS milk. Coverslips were then washed three times in PBS and incubated for 1 h with the appropriate secondary antibody, fluorescein-labeled donkey anti-mouse or rhodamine-labeled donkey anti-rabbit antibody (Jackson ImmunoResearch, West Grove, PA). The coverslips were washed three times with PBS, dipped in sterile distilled water and allowed to dry before mounting with Vectashield with DAPI (Vector Laboratories, Burlingame, CA). All slides were examined using a Zeiss Axioscope II fluorescence microscope (Thornwood, NY) and the images were captured using Cytovision software (Applied Imaging Systems, Santa Clara, CA).
Yeast two-hybrid analysis
The plasmid containing the full-length BRCA2 cDNA was kindly provided by Myriad Genetics (Salt Lake City, UT). Primers were created to amplify amino acid residues 638–1508 containing the BRCA2 BRC repeats 1–3 (BRC1–3) with unique restriction sites BamHI and XhoI and cloned into the pGADT7 vector (Clontech, Palo Alto, CA). Yeast two-hybrid vectors for Rad51 and the paralogs were described previously (Miller et al., 2004). Yeast two-hybrid analysis was performed using the Matchmaker yeast two-hybrid kit as per the manufacturer's instructions (Clontech). AH109 yeast were co-transformed with the BRC1–3 fragment in the activating domain vector and either Rad51, Rad51B, Rad51C, Rad51D, Xrcc2, Xrcc3 or control plasmid in the binding domain vector and assayed for ß-galactosidase activity using o-nitrophenyl-ß-D-galactopyranoside as a substrate, as outlined in the yeast protocols' handbook (Clontech).
EGFP-fusion proteins and direct fluorescence
The full-length Rad51B cDNA was fused to an EGFP by cloning Rad51B from pBacPak9-Rad51B (Miller et al., 2004) in-frame into the EcoRI and PstI restriction enzyme sites in the pEGFP-C2 plasmid (Clontech). The amino acid sequence KKLK, amino acids 4–7 of Rad51B, was mutated to NNLN using the Quikchange site-directed mutatgenesis kit (Qiagen, Valencia, CA) to create the pEGFP-Rad51B/NNLN vector as per the manufacturer's instructions. The pEGFP-Rad51C was created by cloning the Rad51C insert from the pBacPak9-Rad51C plasmid (Miller et al., 2004) into the pEGFP-C2 vector with EcoRI and PstI restriction enzymes. The Xrcc2 cDNA was cloned from the pGBKXrcc2 plasmid (Miller et al., 2004) into the pEGFP-C2 vector using EcoRI and BamHI restriction enzymes. The mRad51D cDNA in Puc19 was obtained from Dr Doug Pittman (Medical College of Ohio, Toledo, OH) and cloned into the pEGFP-C2 vector using EcoRI and BamHI restriction enzymes. All constructs were confirmed by sequencing (Biotech Core, Mountain View, CA). HeLa S3 cells grown on coverslips in 24 well plates were transfected with 1 µg of plasmid DNA using 6 µl of Fugene transfection reagent (Roche Applied Science, Indianapolis, IN) as per the manufacturer's instructions and grown for 48 h. Cells were fixed in 4% formaldehyde in PBS for 30 min and mounted with Vectashield with DAPI (Vector Laboratories). Slides were examined using a Zeiss Axioscope II fluorescence microscope and the images were captured using Cytovision software (Applied Imaging Systems).
Transfection of EGFP-labeled Rad51B in V79 and irs3 cells
Hamster V79 parental and irs3 cells were seeded on glass coverslips in 24 well dishes at a density of 2 x 104 cells/well. Transfection was performed 24 h later in 500 µl of growth medium (without antibiotics) with 2 µl of Lipofectamine 2000 reagent (Invitrogen) and 0.8 µg of DNA. After 24 h, the transfection medium was removed and the cells were fixed with 4% formaldehyde in PBS for 1 h at 4°C. The coverslips were mounted onto glass microscope slides with the addition of 3 µl of Vectashield mounting medium with DAPI (Vector Laboratories). EGFP-labeled protein expression and intracellular localization was examined by fluorescence microscopy. Fluorescence images were captured as described above.
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Nuclear localization of Rad51B
The function of the Rad51 paralogs in the HRR pathway suggests that these proteins might exist primarily in the nucleus of the cell where they interact to repair damaged DNA. Immunostaining with a Rad51B-specific antibody localized the Rad51B protein primarily to the nucleus in HeLa S3 cells as compared with the DAPI-stained controls (Figure 1A and B). Preabsorption of the Rad51B antibody with an epitope-specific peptide abolished this staining indicating that the staining was specific for Rad51B (Figure 1C and D). Immunostaining for Rad51B in MCF10A breast epithelial cells and MCF7 breast cancer cells showed similar Rad51B nuclear localization (data not shown).
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To determine whether Rad51B may contain an independent NLS, an EGFP fusion protein containing full-length Rad51B was examined in HeLa S3 cells by transient transfection. The nuclear localization of the EFGP–Rad51B fusion protein is evidenced by the examination of the direct fluorescence of the EGFP–Rad51B fusion protein in fixed cells in Figure 2B with identical DAPI-stained nuclei in Figure 2E. This suggests that the Rad51B sequence fused to the EGFP provides an NLS that is recognized by the nuclear pore complex and subsequently transported to the nucleus. The EGFP control that did not contain an NLS was found throughout the cell (compare Figure 2A and D, and Figure 2A and B). Sequence analysis of Rad51B revealed a putative NLS in the N-terminus of the protein, amino acids 4–7, KKLK (Rice et al., 1997
). To test the function of this motif, an EGFP–Rad51B mutant, in which the KKLK sequence was mutated to NNLN by site-directed mutagenesis, was transfected into HeLa S3 cells and examined for localization by direct fluorescence. The NNLN mutant was found throughout the cell similar to the EGFP control (compare Figure 2A and C) which suggests that the KKLK motif functions as the NLS of Rad51B.
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Rad51B localizes to the nucleus independently of Rad51C
Nuclear localization of the overexpressed EGFP–Rad51B fusion protein suggests that Rad51B contains it own NLS, as its nuclear import did not seem to rely on any partner protein that may be present in limiting amounts. However, the possibility remained that a partner protein, present in sufficient quantities, might contribute to nuclear localization of endogenous Rad51B. As Rad51B has been shown to bind directly only to Rad51C in the BCDX2 complex, experiments were directed at determining whether Rad51B localized to the nucleus independently or whether it requires the formation of a heterocomplex with Rad51C prior to nuclear import. To address this question, localization of Rad51B was assessed in the V79-derived hamster mutant cell line, irs3, which contains a mutation in exon 6 leading to a Val to Phe amino acid substitution at a highly conserved residue (French et al., 2002
). No Rad51C protein was detected in the irs3 cells, suggesting that this mutation decreases protein stability resulting in a Rad51C-deficient cell line (French et al., 2002). As seen in Figure 3A, the EGFP control construct was localized throughout the transiently transfected cell in comparison with the DAPI-staining in Figure 3D. The results in Figure 3B and E show that the EGFP–Rad51B fusion protein was localized to the nucleus in the Rad51C mutant cell line, demonstrating that absence of Rad51C did not alter Rad51B localization. The EGFP–Rad51B NNLN mutant was observed throughout the irs3 transiently transfected cells (Figure 3C and E), similar to EGFP control and confirming the previous results in the HeLa S3 cells. Similar results to those in irs3 cells were obtained with the V79 parental cell line as a control for Rad51B localization in hamster cells (data not shown). The nuclear localization of Rad51B in Rad51C null cells confirms that Rad51B does not require Rad51C for nuclear localization.
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Rad51B nuclear localization is independent of BRCA2
Nuclear localization of Rad51 has been previously shown to rely on its interaction with BRCA2 (Davies et al., 2001
). The CAPAN-1 pancreatic cancer cell line contains a truncated BRCA2 that mislocalizes BRCA2 to the cytoplasm and eliminates radiation-induced Rad51 nuclear focus formation through sequestration of Rad51 in the cytoplasm (Yuan et al., 1999). We utilized this cell line to determine whether Rad51B, like Rad51, may require a full-length BRCA2 protein for proper localization. As shown in Figure 4A, immunostaining of Rad51B protein was localized to the nucleus in the CAPAN-1 cells, suggesting that it can be transported into the nucleus independently of BRCA2. Merged images from dual labeling for both Rad51B and BRCA2 (Figure 4C) showed no apparent co-localization as the BRCA2 immunostaining was predominantly cytoplasmic and Rad51B immunostaining was primarily nuclear. These results suggest that Rad51B does not require BRCA2 for nuclear localization and, unlike Rad51, Rad51B may not interact with BRCA2.
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Additional experiments were performed to determine whether there is a direct or indirect interaction between Rad51B and BRCA2 in cells containing wild-type BRCA2. HeLa S3 cell extracts were immunoprecipitated with a Rad51B-specific antibody and were examined for co-precipitated BRCA2. This Rad51B-specific antibody has been used to co-precipitate Rad51C from various cell extracts (Miller et al., 2002). Although BRCA2 was present in the extract, BRCA2 did not co-immunoprecipitate with Rad51B (Figure 5A, lanes 1 and 3). Immunoprecipitation of BRCA2 was observed using a Rad51-specific antibody, demonstrating that the immunoprecipitation of the high molecular weight BRCA2 protein was possible from the HeLa S3 cell extracts (Figure 5B, lanes 1 and 3). Preimmune serum did not pull down the BRCA2 protein, demonstrating the specificity of the Rad51–BRCA2 interaction (Figure 5B, lane 2). These data confirm previous results which demonstrated that Rad51 co-precipitates with BRCA2 (Chen et al., 1998a
). Reciprocal co-immunoprecipitations were also performed using a BRCA2-specific antibody to test for complex formation with Rad51 or Rad51B. While a significant amount of Rad51 was co-immunoprecipitated with BRCA2 antibody as compared with an unrelated antibody used as a control, Rad51B was not found to associate with BRCA2 (compare Figure 5C and D, lane 1). Both the Rad51 and Rad51B proteins were detected in the extracts before immunoprecipitation (Figure 5C and D, lane 3). These results suggest that BRCA2 is not found in a complex with Rad51B in vivo.
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To rule out transient or weak interactions that might be undetectable by immunoprecipitation, yeast two-hybrid analysis was used to test for a direct interaction between Rad51B and a fragment of the BRCA2 protein. The BRCA2 protein contains eight BRC repeats. BRC repeats 1–3 (BRC1–3) comprising amino acids 638–1508 of the full-length BRCA2 protein have been previously shown to bind to Rad51 by yeast two-hybrid analysis (Chen et al., 1998a
). This same fragment was tested against the full-length Rad51B protein. Full-length Rad51 was used in the assay as a positive control. The results summarized in Figure 5E show that Rad51 interacts with the BRC1–3 fragment of BRCA2 while no interaction was observed between Rad51B and BRC1–3 in the yeast two-hybrid assay. Moreover, no direct interaction was observed between Rad51C, Rad51D, Xrcc2 or Xrcc3 and this fragment of BRCA2 by yeast two-hybrid analysis, with all the paralog proteins exhibiting binding activity close to that of background levels (Figure 3E).
Subcellular localization of other Rad51 paralogs
Rad51B is found in a heterocomplex with Rad51C as well as in the larger BCDX2 complex in vitro and in vivo (Masson et al., 2001; Liu et al., 2002; Miller et al., 2002; Wiese et al., 2002). Additional EGFP constructs were generated for each of the Rad51 paralog proteins in the BCDX2 complex. As shown in Figure 6A and D, transiently transfected EGFP–Rad51C exhibits a nuclear localization consistent with the recent demonstration that Rad51C contains an NLS at the C-terminal end of the protein (French et al., 2003). The EGFP–Xrcc2 protein was localized in the nucleus as well as the cytoplasm (Figure 6B and E) similar to findings by Liu (2002), in which a GFP–Xrcc2 construct was localized in wild-type V79 cells and found to be not exclusively nuclear in contrast to previous reports (O'Regan et al., 2001). The EGFP–Rad51D also appeared to be localized throughout the transiently transfected cell as compared with the DAPI-stained nuclei (Figure 6C and F), further supporting sequence data that show that Rad51D does not contain an NLS.
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To further characterize the localization of endogenous Rad51C, Rad51D and Xrcc2, HeLa cells were subjected to subcellular fractionation followed by SDS–PAGE and western blot analysis. The results in Figure 7 show that endogenous Rad51C was localized primarily in the nuclear fraction consistent with the finding that it contains an NLS located at the C-terminus (French et al., 2003
). The endogenous Rad51D and Xrcc2 proteins were localized primarily in the nucleus by subcellular fractionation (Figure 7B and C) whereas their EGFP-fusion proteins (Figure 6B and C) appear throughout the cell, suggesting that these proteins may rely on a partner protein that is present in limiting amounts. This limitation could explain why more specific nuclear localization is not seen with the overexpressed EGFP fusions. Further studies are necessary to determine the role of BCDX2 complex formation in the nuclear localization of Rad51D and Xrcc2.
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Using both direct fluorescence and immunocytochemistry, this is the first study that provides direct evidence that Rad51B localizes to the nucleus. A KKLK motif present in the N-terminus of Rad51B appears sufficient to direct the nuclear transport of the Rad51B protein. Moreover, unlike Rad51, the nuclear localization of Rad51B is not affected by the presence of a mutated BRCA2 protein, consistent with the data presented here, demonstrating that there is no direct or indirect association of Rad51B with BRCA2.
It has been suggested through sequence homology that the Rad51 paralogs have diverged from Rad51 in such a way that they would be incapable of mimicking the same binding mode with the BRC repeat (Lo et al., 2003). Sequence alignment of the eight key amino acid residues of Rad51 that interact with the BRC4 of BRCA2 shows at least one polar or charged residue in the paralog sequences that would preclude BRC4 binding to any paralog (Miller et al., 2004). Previously, binding of the Rad51 paralogs has been tested against BRC3, which did not reveal an interaction between BRCA2 and any of the Rad51 paralog proteins (Davies et al., 2001). Our studies examined a larger region of BRCA2 containing BRC motifs 1–3, which demonstrated that Rad51B and the other paralogs did not interact with this region of BRCA2 (Figure 3). It is still possible that the paralogs may interact with the other BRC repeats or a different region of the BRCA2 protein, but it is unlikely that this would be true for Rad51B, given the data presented here. If any of the paralogs does interact with BRCA2, it may be indirectly or under conditions not examined in this study. Our recent domain mapping studies of the BCDX2 complex suggest that the region of Rad51 that binds to BRCA2 may have evolved in the Rad51 paralogs for the purpose of forming unique multimeric complexes such as BCDX2 (Miller et al., 2004).
We have presented evidence that all members of the BCDX2 complex are predominately nuclear; however, to date only Rad51B and Rad51C have been shown to localize independently to the nucleus. We found that EGFP–Rad51C localizes exclusively to the nucleus in HeLa S3 cells, supporting the previous report that Rad51C localizes to the nucleus through its own NLS (French et al., 2003). Rad51D and Xrcc2 co-elute from nuclear extracts of HeLa cells, suggesting that they are present in the nucleus (Kurumizaka et al., 2002). O'Regan showed that the expression of a GFP–Xrcc2 fusion protein is nuclear in the irs1 CHO mutant that is Xrcc2 null (O'Regan et al., 2001). Our preliminary evidence based on EGFP fusions in HeLa S3 cells did not find Rad51D and Xrcc2 to be exclusively nuclear (Figure 6). This result suggests that when GFP–Xrcc2 is expressed in irs1 cells, the fusion protein does not have to compete with endogenous Xrcc2 for binding to a partner protein for nuclear import while in HeLa S3 cells, which contain Xrcc2, the available partner protein is limiting. As sequence analysis does not suggest NLSs in either Rad51D or Xrcc2, we propose that complex formation with Rad51C and Rad51B may occur in the cytoplasm to facilitate nuclear import. Understanding the localization of Rad51 paralog proteins and their interactions will be necessary to fully understand their function in vivo. Further studies will be necessary to determine the possible role of Rad51B in promoting nuclear localization of the BCDX2 complex.
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Acknowledgments |
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The authors would like to thank Francesco Marchetti and Andy Wyrobek for instructions and use of the fluorescence microscope, John Thacker for the irs3 cell line, David Schild for the Rad51 yeast two-hybrid vectors and Doug Pittman for the Rad51D cDNA. This work was supported by an NIH grant R01 #CA 810910 awarded to J.S.A., by an NIH/NCI grant P01 CA92584-02 and by the Low Dose Radiation Research Program, Biological and Environmental Research (BER), US Department of Energy grant awarded to L.H.T. This work was performed under the auspices of the US DOE and the UC–LLNL under Contract no. W-7405-Eng-48.
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Notes |
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* To whom correspondence should be addressed. Tel: +1 925 422 6442; Fax: +1 925 424 6605; Email: albala1{at}llnl.gov
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References |
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Received on September 20, 2004; revised on January 10, 2005; accepted on January 11, 2005.
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