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.
Highlights
The central component in the HR pathway is RAD51, which is the major recombinase in mitotic cells and plays a critical role during meiosis
In addition to BRCA2/PALB2, other important HR mediators are the five RAD51 paralogues (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3), which are required for the assembly of DNA damage-induced RAD51 foci, and cell lines with defects in any of these RAD51 paralogues are defective in HR (14 –16)
We found that the expression of tagged SWI5 or MEI5 was greatly enhanced when they were co-expressed in the cell (Fig. 1B), which indicates that SWI5 and MEI5 form a stable complex in vivo and are mutually interdependent for their stability
Summary
Antibodies—The antibody against RAD51 was described previously [12, 26]. Anti-RPA2 antibody was obtained from. The sequence for RAD51 siRNA was described previously [26, 27]. The siRNA transfection was performed using Oligofectamine (Invitrogen) following the manufacturer’s instructions. Coverslips were washed with PBS and immunostained with primary antibodies in 5% goat serum for 30 min at room temperature. Homologous Recombination Assay—A U2OS cell clone stably expressing HR reporter direct repeat GFP was described previously [28]. This reporter consists of two differentially mutated GFP genes oriented as direct repeats. 2 days after transfection with indicated siRNAs, 1 ϫ 106 U2OS direct repeat GFP cells were electroporated with 20 g of pCBASce, an I-SceI expression vector described previously [29]. Numbers of colonies were counted using a GelDoc with Quantity One software (Bio-Rad)
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