Reconstitution of DNA Strand Exchange Mediated by Rhp51 Recombinase and Two Mediators

Abstract

In the fission yeast Schizosaccharomyces pombe, genetic evidence suggests that two mediators, Rad22 (the S. pombe Rad52 homolog) and the Swi5-Sfr1 complex, participate in a common pathway of Rhp51 (the S. pombe Rad51 homolog)–mediated homologous recombination (HR) and HR repair. Here, we have demonstrated an in vitro reconstitution of the central step of DNA strand exchange during HR. Our system consists entirely of homogeneously purified proteins, including Rhp51, the two mediators, and replication protein A (RPA), which reflects genetic requirements in vivo. Using this system, we present the first robust biochemical evidence that concerted action of the two mediators directs the loading of Rhp51 onto single-stranded DNA (ssDNA) precoated with RPA. Dissection of the reaction reveals that Rad22 overcomes the inhibitory effect of RPA on Rhp51-Swi5-Sfr1–mediated strand exchange. In addition, Rad22 negates the requirement for a strict order of protein addition to the in vitro system. However, despite the presence of Rad22, Swi5-Sfr1 is still essential for strand exchange. Importantly, Rhp51, but neither Rad22 nor the Swi5-Sfr1 mediator, is the factor that displaces RPA from ssDNA. Swi5-Sfr1 stabilizes Rhp51-ssDNA filaments in an ATP-dependent manner, and this stabilization is correlated with activation of Rhp51 for the strand exchange reaction. Rad22 alone cannot activate the Rhp51 presynaptic filament. AMP-PNP, a nonhydrolyzable ATP analog, induces a similar stabilization of Rhp51, but this stabilization is independent of Swi5-Sfr1. However, hydrolysis of ATP is required for processive strand transfer, which results in the formation of a long heteroduplex. Our in vitro reconstitution system has revealed that the two mediators have indispensable, but distinct, roles for mediating Rhp51 loading onto RPA-precoated ssDNA

Expression, purification and crystallization of Swi5 and the Swi5–Sfr1 complex from fission yeast

“…Recent studies have revealed that the Swi5-Sfr1 complex stimulates both Rad51-mediated and Dmc1-mediated strandexchange reactions by activating the recombinases directly. Thus, Swi5-Sfr1 acts as an activator of the recombinases (Haruta et al, 2007;Kurokawa et al, 2008). Determining the crystal structures of Swi5 and Sfr1 is of importance because such molecular information will provide a better understanding of the function of Swi5 and Sfr1 in homologous recombination.…”

The fission yeast meiosis-specific Dmc1 recombinase mediates formation and branch migration of Holliday junctions by preferentially promoting strand exchange in a direction opposite to that of Rad51

“…The Swi5-Sfr1 complex is an auxiliary protein for both Rad51 and Dmc1 (Akamatsu et al 2003(Akamatsu et al , 2007Ellermeier et al 2004). We demonstrated that Swi5-Sfr1 stimulates three-strand exchanges mediated in vitro by Rad51 and Dmc1 (Haruta et al 2006;Kurokawa et al 2008). We also discovered ) that both fission yeast and human Rad51 proteins can promote the formation and BM of HJs, with a four-strand exchange assay containing gDNA and its homologous ldsDNA (Supplemental Fig.…”

“…1B). Swi5-Sfr1 complex and RPA were added to the reaction, both of which have been shown to stimulate the three-strand exchange reaction mediated by Rad51 or Dmc1 (Haruta et al 2006;Kurokawa et al 2008). In both 39-end and 59-end homology reactions,similar high amounts of JMs-especially fJMs corresponding to the s structure-were formed within 30 min after reaction initiation (Fig.…”

“…Expression and purification of Dmc1, Swi5-Sfr1, Rad51, and RPA from fission yeast and RecA and RuvC from E. coli were essentially as described (Haruta et al 2006;Kurokawa et al 2008;Murayama et al 2008). Human RPA was expressed in E. coli and was purified essentially as described (Henricksen et al 1994).…”

Dual regulation of Dmc1-driven DNA strand exchange by Swi5–Sfr1 activation and Rad22 inhibition

“…These complexes are thought to stabilize and/or activate Rad51 filaments to promote the strand exchange reaction (Akamatsu et al 2003(Akamatsu et al , 2007Ellermeier et al 2004;Kurokawa et al 2008). Notably, the Swi5-Sfr1 complex exhibits very low mediator activity (i.e., this complex does not significantly help Rad51 to bind ssDNA that is already coated with RPA), but additional information on this complex, which is required for full levels of recombination (Akamatsu et al 2003;Ellermeier et al 2004), is lacking, although its sharply kinked structure has been determined by X-ray crystallography (Kuwabara et al 2012).…”