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. 2011 Mar 18;286(11):9888-93.
doi: 10.1074/jbc.M110.207290. Epub 2011 Jan 20.

The Role of the Human SWI5-MEI5 Complex in Homologous Recombination Repair

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Free PMC article

The Role of the Human SWI5-MEI5 Complex in Homologous Recombination Repair

Jingsong Yuan et al. J Biol Chem. .
Free PMC article

Abstract

The Swi5-Mei5 complex and its homologues are involved in specialized recombination pathways in budding and fission yeasts. Although the fission yeast homologue Swi5-Sfr1 is critical for homologous recombination repair, the budding yeast counterpart Sae3-Mei5 is meiosis-specific, interacts with Dmc1, and promotes assembly of Dmc1 on meiotic chromosomes. Here, we identify and characterize the human SWI5-MEI5 (C9orf119-C10orf78) complex. We showed that SWI5 and MEI5 form a stable complex in vitro and in vivo. The C-terminal Swi5 domain of SWI5 and the middle coiled-coil region of MEI5 dictate this conserved interaction. In addition, SWI5-MEI5 directly interacts with RAD51 in vitro. Depletion of SWI5 or MEI5 in human cells causes defects in homologous recombination repair. Finally, SWI5- or MEI5-depleted cells display enhanced sensitivity to ionizing radiation, consistent with the role of this complex in HR repair. Our results suggest that human SWI5-MEI5 has an evolutionarily conserved function in homologous recombination repair.

Figures

FIGURE 1.
FIGURE 1.
SWI5 and MEI5 form an evolutionarily conserved protein complex. A, all SWI5 or MEI5 orthologues contain a Swi5 or Mei5 domain. Conserved coiled-coil motifs (CC) are indicated within both Swi5 and Mei5 domains. The accession numbers of SWI5 orthologues are: NP_001035100.1 for Homo sapiens, XP_001144446.1 for Pan troglodytes, NP_780399.1 for Mus musculus, XP_575112.2 for Rattus norvegicus, XP_002663552.1 for Danio rerio, NP_001107337.1 for Xenopus (Silurana) tropicalis, NP_595453.1 for Schizosaccharomyces pombe, and NP_011947.2 for Saccharomyces cerevisiae. The accession numbers of MEI5 orthologues are: NP_660290.3 for H. sapiens, XP_001135759.1 for P. troglodytes, NP_080653.2 for M. musculus, NP_001041321.1 for R. norvegicus, NP_001076329.1 for D. rerio, XP_002936845.1 for X. (Silurana) tropicalis, NP_595668.1 for S. pombe, and NP_015204.1 for S. cerevisiae. B, the interaction between SWI5 and MEI5 was confirmed by co-immunoprecipitation (IP) experiments. 293T cells were transfected with plasmids encoding Myc-tagged SWI5 or MEI5 together with plasmids encoding SFB-tagged MEI5 or SWI5 as indicated. SFB-tagged C1orf57 was used here as a control. C, SWI5 and MEI5 directly interact with each other. MBP-tagged and GST-tagged proteins were expressed and purified from E. coli. Pulldown assays were performed by incubating purified proteins together with glutathione-agarose beads in NETN buffer. Beads were washed, associated proteins were eluted and detected by Coomassie staining and immunoblotting using antibodies as indicated.
FIGURE 2.
FIGURE 2.
The C-terminal Swi5 domain of SWI5 and the middle coiled-coil region of MEI5 mediate the formation of this protein complex. A, shown is a schematic representation of full-length SWI5 and the mutants used in this study. Their abilities to bind to MEI5 are indicated. FL, full-length. B, the binding of SWI5 to MEI5 is greatly impaired with the deletion of the C-terminal Swi5 domain. C, a point mutation (L173P) within the coiled-coil motif of SWI5 does not affect its binding to MEI5. D, shown is a schematic representation of full-length MEI5 and the mutants used in this study. Their SWI5-binding abilities are indicated. E, a fragment containing coiled-coil motif of MEI5 is required for the binding of MEI5 to SWI5. 293T cells were transfected with plasmids encoding Myc-tagged and SFB-tagged proteins. Co-immunoprecipitation (IP) was performed as described under “Experimental Procedures.”
FIGURE 3.
FIGURE 3.
SWI5-MEI5 directly interacts with RAD51 and is involved in homologous recombination repair. A, SWI5 and MEI5, especially SWI5, can directly bind to RAD51. B, the efficiency of down-regulating the expression of their target genes by various siRNAs was evaluated by RT-PCR. C, down-regulation of SWI5 or MEI5 greatly impairs IR-induced RAD51 foci formation without influencing RPA foci formation. Immunostaining was performed 6 h after IR treatment with the indicated antibodies. D, decreasing of SWI5 or MEI5 impairs HR repair. U2OS direct repeat GFP cells were electroporated with pCBASce plasmids (see “Experimental Procedures” for details). The percentage of GFP-positive cells was determined by flow cytometry 48 h after electroporation. The data were normalized to those obtained from cells transfected with control siRNA (set as 1.0). Means and S.E. (error bars) shown are obtained from three independent experiments. E, cells with SWI5 or MEI5 down-regulation display increased IR sensitivity. Cell survival assays were performed as described under “Experimental Procedures.” Data were presented as means and S.D. (error bars) from three different experiments. Ctrl, control; Gy, gray.

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