Mol. Hum. Reprod. Advance Access originally published online on October 15, 2004
Molecular Human Reproduction 2004 10(12):917-924; doi:10.1093/molehr/gah123
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Association between MSH4 (MutS homologue 4) and the DNA strand-exchange RAD51 and DMC1 proteins during mammalian meiosis
1FRE 2720 CNRS/UNSA, Equipe M3R, LRC CEA No. 32-V, 2U 145 INSERM, Faculté de Médecine, Av. de Valombrose, 06107 Nice Cedex 2, France and 3Department of Biology, York University, 4700 Keele Street, Ontario, Canada, M3J1P3
4 To whom correspondence should be addressed at: FRE 2720 CNRS/UNSA, Faculté de Médecine, Avenue de Valombrose, 06107 Nice Cedex 2, France. Email: santucci{at}hermes.unice.fr
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Abstract |
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During meiotic prophase, chromosomes must undergo highly regulated recombination events, some of which lead to reciprocal exchanges. In yeast, MSH4, a meiosis-specific homologue of the bacterial MutS protein, is required for meiotic recombination. In mice, disruption of the Msh4 gene results in male and female infertility due to meiotic failure. To date, the implication of MSH4 mutations has not been established in human sterility. However, it is noteworthy that mutant mice exhibit a defect in the chromosome synapsis, strikingly similar to the clinical observations found in human infertility. As a step towards understanding the molecular mechanisms underlying the role of MSH4 in human gametogenesis, we decided to determine whether this protein interacts with recombination machinery enzymes. Our results provide biochemical evidence indicating that the human MSH4 protein physically interacts with both RAD51 and DMC1, two RecA homologues known to initiate DNA strand-exchange between homologous chromosomes. Immunolocalization analyses show that some MSH4 foci, located on mouse meiotic chromosomes, colocalize with DMC1/RAD51 complexes. Our data support the view that MSH4 is associated with the early meiotic recombination machinery in mammals. We consider the possibility that MSH4 is involved in the regulation of recombination events by exerting a function closely after DNA strand-exchange has been initiated.
Key words: DNA strand-exchange protein/infertility/meiosis/MSH4/recombination
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Introduction |
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In mammals, as in most organisms, two prominent features of meiotic prophase are juxtaposition (synapsis) of homologous chromosomes along a specialized proteinaceous structure, the synaptonemal complex (SC) and genetic recombination between homologous chromosomes. Reciprocal recombination events (crossovers) establish chiasmata, which are physical connections between homologues that ensure their correct segregation at first meiotic division. The MSH4 protein, a meiosis-specific homologue of the bacterial MutS mismatch repair protein, plays no role in mismatch correction; instead, in budding yeast and nematode, this protein is required for wild-type level of meiotic crossovers (Ross-MacDonald and Roeder, 1994
; Zalevsky et al., 1999). MSH4 has been proposed to function in stabilization and/or resolution of late meiotic recombination intermediates. However, recent findings indicate that MSH4 may be involved earlier in yeast meiotic prophase than initially proposed; MSH4 localizes to sites of synapsis initiation and msh4 yeast mutants exhibit not only a low level of crossovers, but also a defect in chromosome synapsis (Novak et al., 2001). In mammals, immunolocalization on meiotic chromosomes has led to the hypothesis that the MSH4 protein is involved in successive steps of meiotic prophase (Santucci-Darmanin et al., 2000). First, MSH4 is present at sites along the SC, as soon as homologues synapse during zygotene stage, suggesting that this protein acts in synaptic events. Consistent with this proposal, in MSH4 homozygous mutant (Msh4 –/–) mice, germ cells exhibit a dramatic defect in chromosome synapsis (Kneitz et al., 2000). These cells do not progress beyond the zygotene, resulting in sterility. Second, we have previously shown that MSH4 interacts with MLH1 and MLH3 (two homologues of the bacterial MutL protein) and that MSH4 and MLH1 colocalize on mouse meiotic chromosomes at a later stage of meiotic prophase (mid-pachytene) (Santucci-Darmanin et al., 2000, 2002). Examination of mutant mice indicates that MLH1 and MLH3 are not required for synapsis, but that these two proteins are involved later in meiotic prophase in the formation of meiotic crossover products (Baker et al., 1996; Edelmann et al., 1996; Lipkin et al., 2002). Taken together, these results suggest that, in mammals, MSH4 acts first, without MLH1 and MLH3 at the step of synapsis and, in ensuing steps, in conjunction with these two MutL homologues to promote crossovers.
The relationship between synapsis and homologous recombination events is not well established in mammals. However, many lines of evidence, such as the phenotypes of mice carrying disruption of meiotic genes, indicate the importance of the recombination process in promoting chromosome synapsis. In mammals, as in yeast, Spo11, a type II topoisomerase, seems to have a central role in meiosis by generating the DNA double-strand breaks (DSBs) that initiate meiotic recombination (Keeney et al., 1997; Mahadevaiah et al., 2001). Spo11 homozygous mutant mice display chromosome synapsis defects, thereby providing evidence that the initiation of recombination precedes and is required for normal synapsis (Baudat et al., 2000; Romanienko and Camerini-Otero, 2000). Based on genetic analyses in yeast and biochemical studies, DMC1 and RAD51, two homologues of Escherichia coli strand transfer enzyme RecA, appear to be involved in the subsequent processing of the resected DSBs by promoting strand exchange between homologous chromosomes (Baumann et al., 1996; Li et al., 1997; Schwacha and Kleckner, 1997). RAD51 is expressed in both mitotic and meiotic cells, whereas DMC1 is meiosis-specific (Shinohara and Ogawa, 1999). Consistent with the role of RAD51 and DMC1 in the early steps of the meiotic recombination process, these two proteins colocalize on mouse meiotic chromosomes at leptonema prior to any synapsis (Tarsounas et al., 1999). They continue to be observed during zygonema and early pachynema. The Rad51 null mutation is lethal during mouse embryogenesis (Lim and Hasty, 1996; Tsuzuki et al., 1996), while Dmc1 null mutation causes meiotic arrest associated with abnormal synapsis (Pittman et al., 1998; Yoshida et al., 1998). Taken together, these results support the view that strand exchange between homologous chromosomes is required for a correct synapsis.
In mammals, the molecular mechanisms regulating temporal progression through meiosis are still unknown. For that reason, the identification of factors causing human infertility is impaired. The phenotype of Msh4 mutant mice and MSH4 immunolocalization on meiotic chromosomes suggest that MSH4 has an early function in mammalian meiotic recombination. Aberrations in meiotic chromosome behaviour in Msh4 –/– mice correspond closely to defects observed in Spo11-deficient and DMC1-deficient mice. In order to understand the role of MSH4 during mammalian meiosis, we tested whether this protein interacts with recombination machinery enzymes. Here, we show a physical interaction, both in vivo and in vitro, between the human MSH4 protein and the RecA homologues, RAD51 and DMC1.
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Antibodies
Goat polyclonal anti-GST, mouse monoclonal anti-His tag and goat polyclonal anti-His tag antibodies were obtained from Amersham Biosciences (Uppsla, Sweden), Qiagen (Hilden, Germany) and Abcam (Cambridge, UK), respectively. Rabbit polyclonal anti-RAD51 and mouse monoclonal anti-DMC1 antibodies, used in Western blotting experiments, were obtained from Merck-Eurolab (Spoenga, Sweden) and Abcam, respectively. The rabbit polyclonal anti-MSH4 antibodies, previously described (Santucci-Darmanin et al., 2000
), recognize both human and mouse MSH4 proteins. Secondary antibodies used in Western blotting experiments were goat anti-rabbit immunoglobulin G (IgG) (Amersham Biosciences), goat anti-mouse IgG (Amersham Biosciences) and rabbit anti-goat IgG (Dako, Carpinteria, CA, USA) conjugated with horse-radish peroxidase. The mouse anti-DMC1 antibody used for immunocytology has been previously described (Freire et al., 1998). This antibody cross-reacts with RAD51 unless immunodepleted for cross-reaction epitopes. In a study with epitope-specific, immunodepleted DMC1 and RAD51, Tarsounas et al. (1999) observed that these two proteins completely colocalize when observed with immunofluorescence and electron microscopy. Therefore, the mouse anti-DMC1 used in this study allows the detection of DMC1/RAD51 complexes. The anti-Cor1 antibodies generated in mice and the CREST anti-centromere serum have also been previously described (Dobson et al., 1994).
Plasmid construction
All primer sequences used for PCR amplification are shown in Table I. A two-step PCR procedure was used to amplify the RAD51, DMC1 and MSH5 coding sequences from human testis cDNAs (Human Testis Quick-clone cDNA, BD Biosciences, San Jose, CA, USA). Primers used to amplify RAD51 cDNA were Rad51a plus Rad51b and nested Rad51c plus Rad51d, which included a BamHI and a SmaI restriction site, respectively. RAD51 coding sequence was digested and cloned between BamHI and SmaI sites of either pET-28a (Novagen, Madison, WI, USA) or pGEX-4T2 (Amersham Biosciences) vectors to generate pET-hRAD51 and pGEX-hRAD51, respectively. DMC1 cDNA was amplified by using Dmc1a plus Dmc1b and nested Dmc1c plus Dmc1d primers. Dmc1c and Dmc1d included BamHI and XhoI sites, respectively. Amplified DMC1 cDNA was digested and introduced between BamHI and XhoI of either pET-28a or pGEX-4T2 to yield pET-hDMC1 and pGEX-hDMC1, respectively. To obtain MSH5 cDNA, amplifications were performed by using primers Msh5a plus Msh5b and nested Msh5c plus Msh5d primers which harbour a BamHI and XhoI restriction site, respectively. Amplified MSH5 coding sequence was digested with BamHI and XhoI and ligated between the two corresponding sites into the pET-28a vector yielding the pET-hMSH5 construct. The pGEX-hMSH4 and pCR3.1-hMSH4 vectors have been previously described (Santucci-Darmanin et al., 2002). For the yeast two-hybrid assay, the human MSH4 cDNA was amplified by PCR with Msh4a and Msh4b primers (which contain a XmaI and SalI site, respectively), using pGEX-hMSH4 plasmid as template. PCR products were digested with XmaI and SalI and cloned into pBTM116 (Vojtek et al., 1993) as fusion with the LexA DNA binding domain (pBTM-hMSH4). Human RAD51 and DMC1 coding sequences were PCR-amplified with Rad51e/Rad51f and Dmc1e/Dmc1f primers, using pGEX-hRAD51 and pGEX-hDMC1 as template, respectively. Amplified RAD51 and DMC1 cDNAs were digested by NcoI/XmaI and BamHI/XhoI, respectively, and ligated into the two-hybrid vector pAct2 (Clontech, Palo Alto, CA, USA) as fusion with Gal4 activation domain (AD) to yield pAct-hRAD51 and pAct-hDMC1 plasmids. pAct-hMSH5 was a gift from R. Kolodner. All constructs were verified by DNA sequencing.
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Yeast two-hybrid assays
Yeast two-hybrid constructs were transformed into Saccharomyces cerevisiae strain L40 (Mat a, his3-200, trp1-901, leu2-3112, ade2, LYS2 :: (lexA op)4-HIS3, URA3 :: (lexA op)8-lacZ GAL4) using the lithium acetate method. Yeast transformants harbouring both DNA binding domain and AD constructs were selected on SD/-Leu-Trp medium. Histidine prototrophy was assayed on SD/-Leu-Trp-His medium containing 1 mM 3-amino-1,2,4-triazole (3-AT). ß-Galactosidase activities were qualitatively monitored by the blue colour development with the X-gal filter assays.
In vitro translation
pCR3.1-hMSH4, pET-hRAD51, pET-hDMC1 and pET-hMSH5 were added to coupled transcription–translation rabbit reticulocyte lysates (Promega, Madison, WI, USA) with [35S]L-methionine (Amersham Biosciences), according to the manufacturer's instructions.
Expression of GST-fusion proteins
Escherichia coli BL21 (DE3) (Stratagene, Cedar Creek, TX, USA) cells were transformed with each one of the pGEX constructs. Fresh overnight cultures of transformed cells were diluted 1 in 100 with Lurian broth medium and ampicillin. After growth at 37°C to an A600 of 0.6, protein expression was induced with 0.1 mM IPTG (isopropyl-1-thio-ß-D-galactopyranoside) for 75 min at 30°C. In order to check the expression of fusion proteins, whole-cell lysates were prepared and submitted to immunoblotting analysis by using the following antibodies: goat polyclonal anti-GST (1:5000), rabbit polyclonal anti-hMSH4 (1:2000), rabbit polyclonal anti-hRAD51 (1:2000) and mouse monoclonal anti-hDMC1 (1:2000).
GST-fusion protein interaction assay with in vitro translated products
Fresh pellets of induced bacterial cells expressing the various glutathione-S-transferase (GST)-fusion proteins were used to prepare lysates as follows. The pellet was resuspended in ice-cold phosphate-buffer saline (PBS) with complete EDTA-free protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) (1 volume buffer per 60 volume bacterial culture). A 10-min digestion on ice with 1 mg/ml lysozyme followed. After addition of 0.2% Triton X-100 and 1 mM dithiothreitol (DTT), the suspension was snap-frozen in liquid nitrogen and thawed twice. Resulting lysates were incubated with DNase I (200 U/ml) for 40 min on ice and centrifuged at 10 000 g for 30 min at 4°C. The glutathione-S-Sepharose 4B beads (Amersham-Pharmacia) were added to lysate, so that 
1 µg of GST-fusion proteins were bound to 50 µl of beads. After incubation for 2 h at 4°C under gentle agitation, the coated GST-fusion protein beads were washed twice in buffer A: 50 mM Tris–HCl, pH 8, 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% sodium deoxycholate, 2 mM DTT with complete EDTA-free protease inhibitor cocktail, then washed once in buffer A adjusted to 1 M NaCl, once again in buffer A and twice in buffer B: 20 mM Tris–HCl pH 8, 150 mM NaCl, 0.1% Nonidet P40, 10% glycerol, 750 µg/ml bovine serum albumin (BSA), 5 mM EDTA and 2 mM DTT with complete protease inhibitor cocktail. The binding reaction was performed in 1 ml of buffer B, with 50 µl of coated GST-fusion protein beads and different volumes of in vitro translated products (either 7.5 µl of hMSH4, 5 µl of hMSH5, 7.5 µl of hRAD51 or 7.5 µl of hDMC1, so that equal amounts of radiolabelled proteins were used in each assay). After 1 h of incubation at 4°C under gentle agitation, the bound complexes were sedimented by centrifugation and washed as follows: twice in buffer B, once in buffer B adjusted to 1 M NaCl and twice again in buffer B. Then, the beads were resuspended in 50 µl of SDS-PAGE loading buffer and heated for 5 min at 95°C. Protein samples were resolved on an 8% SDS-PAGE. Gels were fixed, treated with Amplify (Amersham Biosciences) and dried before exposure at –70°C.
Immunoprecipitations
Co-immunoprecipitation analysis of hMSH4/hRAD51 and hMSH4/hDMC1 interactions was carried out by using bacterial expressed recombinant fusion proteins. BL21 (DE3) bacterial cells were doubly transformed with pGEX-hMSH4 and pET-hRAD51 or pGEX-hMSH4 and pET-hDMC1 in order to allow co-expression of GST-hMSH4 with either His6-hRAD51 or His6-hDMC1, respectively. Induction of protein expression and bacterial cells lysis procedure were the same as described for GST-fusion protein interaction assay. Cell lysates were then incubated overnight at 4°C with either 5 µg of mouse monoclonal Penta.His antibody or 5 µg of anti-GST goat polyclonal antibodies. After addition of 50 µl of a 50% slurry of BSA-saturated protein-A-Sepharose beads (Amersham Biosciences), for 1 h at 4°C, immunoprecipitates were recovered by centrifugation and washed five times by using PBS supplemented with 300 mM NaCl, 0.2% Triton X-100, 1 mM DTT and with complete EDTA-free protease inhibitor cocktail. Immunoprecipitated proteins were submitted to Western blotting analysis by using indicated antibodies.
Immunocytology
Mouse testicular cells were surface-spread on 0.1 M NaCl, attached to glass slides, fixed in 2% paraformaldehyde with 0.03% SDS, washed in 0.4% Kodak Photo-Flo 200 and dried briefly. After washes in 0.1% Triton X-100 and 10% ADB (antibody dilution buffer: 10% goat serum, 3% BSA, 0.05% Triton X-100) in PBS, nuclei were incubated in primary antibodies (1:500 dilution in ADB in PBS), overnight at room temperature. After washes, nuclei were incubated in fluorescein isothiocyanate (FITC) and/or rhodamine-conjugated secondary antibodies (1:500 dilution in ADB in PBS) for 1 h at 37°C. After washing and drying, the slides were mounted in ProLong antifade with 4 µg/ml DAPI. Images were recorded on Fujichrome ASA 400 film, scanned, recorded on disk and printed on an Epson 870 printer. For electron microscopy, plastic-coated glass slides were used. Spermatocyte nuclei became attached to the thin plastic carrier and were fixed as described for immunofluorescence analysis. Immune-staining was performed by using gold-conjugated secondary antibodies (5, 10 and 15 nm gold grains). Then, the plastic film was floated off and transferred to nickel EM grids. Electron density of the chromosome cores and SCs was increased by staining with 1% osmium tetroxide. Electron micrographs of cores/SCs and immunogold were recorded at various magnifications and were photographically enlarged.
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Results |
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hMSH4 interacts with hRAD51 and hDMC1 in a yeast two-hybrid assay
We have used the yeast two-hybrid system to test for direct interaction of the hRAD51 and hDMC1 proteins with hMSH4. The hRAD51 and hDMC1 coding sequences were cloned into the AD vector and hMSH4 open reading frame was introduced into the DNA binding domain (BD) vector. The ß-galactosidase activities were obtained when hMSH4-BD was co-expressed in the L40 yeast strain with either hRAD51-AD or hDMC1-AD (Figure 1), suggesting that hMSH4 associates with the two RecA homologues. A separate reporter gene HIS3 was also used to validate observed protein interactions. Histidine prototrophy phenotypes were ascertained from L40 double transformants harbouring the same pairs of two-hybrid constructs that could lead to LacZ activation. As positive control, we have analysed the reporter gene activation for a hMSH5–hMSH4 protein pair, which is a previously characterized interaction (Bocker et al., 1999
). We failed to observe the LacZ and the HIS3 gene activation in yeast double transformants expressing only one of the two testing proteins.
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hMSH4 binds RecA homologues in vitro
The interaction of hMSH4 with hRAD51 and hDMC1 was confirmed in vitro through GST pull-down assays. Pull-down experiments were carried out by using the GST-fusion proteins expressed in E. coli and [35S]-radiolabelled in vitro transcribed and translated (IVTT) proteins. GST-hMSH4 and GST proteins were purified from bacterial cell lysates, immobilized onto glutathione-Sepharose beads and these matrices were incubated with either IVTT-hRAD51, IVTT-hDMC1 or IVTT-hMSH5 (positive control). As shown in Figure 2A, GST-hMSH4, but not GST alone, resulted in retention of IVTT-hRAD51 and IVTT-hDMC1 proteins. In reciprocal experiments (Figure 2B), IVTT-hMSH4 was shown to precipitate with GST-hRAD51 and GST-hDMC1. The direct interaction between hRAD51 and hDMC1, which has been previously described (Masson et al., 1999
), was used as positive control. Consistent with the results of the two-hybrid analysis, this in vitro binding assay demonstrates that the recombinant hMSH4 protein can specifically interact with the recombinant hRAD51 and hDMC1 proteins.
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Co-immunoprecipitation of hMSH4 with hRAD51 and hDMC1
We then tested whether this MutS homologue could be co-immunoprecipitated with either hRAD51 or hDMC1. For this purpose, the recombinant GST-hMSH4 protein was co-expressed in E. coli BL21 (DE3) with either His6-hRAD51 or His6-hDMC1. Lysates prepared from these bacterial cells were submitted to immunoprecipitation with a monoclonal anti-His tag antibody. As control, lysates collected from bacterial cells harbouring the pGEX-hMSH4 and the empty pET-28a plasmids were incubated with the same antibody. Immunoprecipitates were analysed by Western blotting, with either rabbit polyclonal anti-hMSH4 or goat polyclonal anti-His tag antibodies (Figure 3A). As expected, the monoclonal anti-His tag antibody allowed immunoprecipitation of the His6-hRAD51 and His6-hDMC1 proteins. GST-hMSH4 was found to co-immunoprecipitate with either His6-hRAD51 or His6-hDMC1. Immunoprecipitation of GST-hMSH4 was entirely dependent on the expression of the recombinant RecA homologues. In reciprocal experiments (Figure 3B), each one of the RecA homologues was found with GST-hMSH4 in immunoprecipitates obtained with anti-GST antibodies and anti-GST antibodies could precipitate His6-hRAD51 or His6-hDMC1 only when the recombinant hMSH4 protein was present in lysates. These results indicate that hMSH4 binds hRAD51 and hDMC1 in soluble cell extracts.
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Colocalization of MSH4 with the RecA homologues on mouse meiotic chromosomes
We next tested for the ability of MSH4 to colocalize with RAD51 and DMC1 on mouse meiotic chromosomes. Previous analyses have shown that DMC1/RAD51 complexes are associated with unpaired chromosome cores at the leptotene stage (Tarsounas et al., 1999
). Thereafter, the number of DMC1/RAD51 foci declines from zygotene to pachytene. In contrast, MSH4 is not associated with unpaired chromosomes. MSH4 foci appear at zygotene and remain along SCs during early pachytene, then the number of MSH4 foci decreases until late pachytene (Santucci-Darmanin et al., 2000). For the present study, mouse spermatocyte nuclei were doubly labelled with red rhodamine for DMC1 antigen and with green fluorescent FITC for the MSH4 antigen to permit a comparative study of the DMC1/RAD51 and the MSH4 protein, relative to each other. Figure 4 shows a pachytene spermatocyte nucleus doubly labelled; we observed green (Figure 4A and D) and red foci (Figure 4B and E) corresponding to MSH4 protein and DMC1/RAD51 complexes, respectively. On the merged image (Figure 4C and FFigure 4C and F), yellow foci that mark sites where MSH4 and DMC1/RAD51 complexes colocalize were observed. The images of green MSH4 foci and red DMC1/RAD51 foci are slightly offset to better illustrate examples of colocating foci in Figure 4G. This pachytene nucleus has 118 MSH4 foci, 67 autosomal DMC1/RAD51 foci and 12 mixed MSH4-DMC1/RAD51 foci. A bar graph (Figure 5) illustrates the counts performed from immunofluorescence labelling of seven nuclei at different stages ranging from mid-zygotene to mid-pachytene. From these analyses, it appeared that mixed MSH4-DMC1/RAD51 foci are present, on synapsed chromosome cores, during these successive stages of meiotic prophase, although these mixed foci are in minority at all times, as previously described (Moens et al., 2002). Association between MSH4 and DMC1/RAD51 foci was also observed when we used immunoelectron microscopy, which allows analysis of protein localization at higher resolution. In Figure 6A, we observed groups of 10 nm grains that mark the sites of DMC1/RAD51 antigens on synapsed chromosome cores. Several DMC1/RAD51 clusters do not contain detectable 5 nm grains, corresponding to MSH4 antigen. Nevertheless, a group of 10 nm grains is in close contact with a few 5 nm grains, establishing the colocalization of MSH4 with DMC1/RAD51 complexes. This result was confirmed by a higher magnification of the MSH4 and DMC1/RAD51 proteins on synapsed chromosome cores (Figure 6B). Consistent with interaction data, these immunocytological analyses show a partial colocalization of MSH4 and DMC1/RAD51 foci on mouse meiotic chromosomes when observed with fluorescence or electron microscopy.
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Discussion |
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Here, we provide evidence that MSH4, in mammals, associates with the early meiotic recombination machinery. Indeed, we show physical interactions between the human MSH4 protein and the two strand-exchange proteins, RAD51 and DMC1. These interactions were detected using a two-hybrid system, in vitro binding assays and co-immunoprecipitation experiments with bacterially expressed proteins. In agreement with the interaction data, cytological analyses show a colocalization of MSH4 and DMC1/RAD51 complexes at some foci on mouse meiotic chromosome SCs. The degree of colocalization observed between MSH4 and RecA homologues is consistent with the fact that immunoprecipitation of RAD51 and DMC1 from mouse spermatocyte extracts does not routinely pull-down the MSH4 protein (Neyton, unpublished observations).
RAD51 and DMC1 are supposed to function in conjunction during mammalian meiotic prophase to promote strand exchange and homology search between homologous chromosomes. Whereas DMC1/RAD51 are seen as soon as leptotene associates with unpaired chromosome cores (Tarsounas et al., 1999), MSH4 appears only at the zygotene stage when homologous chromosomes begin to synapse (Santucci-Darmanin et al., 2000). The late appearance of MSH4 relative to the strand-exchange proteins suggests that MSH4 may function after joint molecule formation between homologous chromosomes. We observed foci on mouse meiotic chromosome cores where MSH4 protein and DMC1/RAD51 complexes are in close contact. These observations suggest that MSH4 may join some DMC1/RAD51 foci and interact with this complex in order to exert a function, closely after joint molecule formation. However, the degree of colocalization between MSH4 and the RecA homologues and the fact that MSH4 foci persist, whereas DMC1/RAD51 complexes are lost at the onset of pachynema, suggest that MSH4 may exert functions independent of the DNA strand-exchange proteins.
Biological significance of the interaction between MSH4 and RecA homologues remains to be determined. In most meiotic species, it is now established that crossover distribution along and among meiotic chromosomes is highly regulated; crossovers are not randomly distributed, property known as crossover interference (Roeder, 1997). Thus, only a subset of early recombinational interactions at the DNA level is specifically designated to mature as crossovers. The mechanism of this crossover control process remains to be defined. However, genetic analyses have led to the conclusion that the budding yeast MSH4 protein functions in the regulation of crossover distribution (Khazanehdari and Borts, 2000; Novak et al., 2001). Furthermore, recent studies in yeast (reviewed by Bishop and Zickler, 2004) suggest that the crossover versus non-crossover decision is made very early in the meiotic recombination process, most probably prior to or during stable DNA strand-exchange formation, between homologous chromosomes. Whether MSH4 is involved in crossover interference in mammals is unclear. Nevertheless, it is interesting to speculate that the interaction between MSH4 and DMC1/RAD51 proteins could be involved in the selection of early DNA–DNA interactions as future crossover sites. One can assume that MSH4 could interact with RecA homologues closely after the strand invasion step in order to induce or maintain a particular geometry of strand invasion that would influence the crossover outcome.
Our data suggest that MSH4 may interact with the early recombination machinery during mammalian meiotic prophase. Further biochemical and immunological analyses are required to understand the biological significance of these interactions. Disruption of several MutS and MutL homologues in mouse has demonstrated a critical role of these genes in meiotic recombination. These mouse models suggest that some of the MutS and MutL homologues could be candidates for human infertility. Therefore, it will be of great interest to conduct further studies in order to characterize the mechanism of MutS and MutL-like proteins participating in mammalian meiotic recombination.
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Acknowledgements |
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We are grateful to R. Kolodner for kindly providing the pAct-hMSH5 plasmid and to F. Lahaye and L. Palin for technical assistance. This work was supported by grants, to V. P-F, from the Association pour la Recherche contre le Cancer (ARC No. 5115) and the Commissariat à l'Energie Atomique (LRC CEA No. 32-V). S.N. is supported by a fellowship from the Ligue contre le Cancer.
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References |
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Submitted on July 16, 2004; resubmitted on September 29, 2004; accepted on October 4, 2004.
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