Functional Interactions among Yeast Rad51 Recombinase, Rad52 Mediator, and Replication Protein A in DNA Strand Exchange

Binwei Song,Patrick Sung

doi:10.1074/jbc.m910244199

Binwei Song, Patrick Sung

Open Access

https://doi.org/10.1074/jbc.m910244199

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Abstract

Rad51-catalyzed DNA strand exchange is greatly enhanced by the single-stranded (ss) DNA binding factor RPA if the latter is introduced after Rad51 has already nucleated onto the initiating ssDNA substrate. Paradoxically, co-addition of RPA with Rad51 to the ssDNA to mimic the in vivo situation diminishes the level of strand exchange, revealing competition between RPA and Rad51 for binding sites on ssDNA. Rad52 promotes strand exchange but only when there is a need for Rad51 to compete with RPA for loading onto ssDNA. Rad52 is multimeric, binds ssDNA, and targets Rad51 to ssDNA. Maximal restoration of pairing and strand exchange requires amounts of Rad52 substoichiometric to Rad51 and involves a stable, equimolar complex between Rad51 and Rad52. The Rad51-Rad52 complex efficiently utilizes a ssDNA template saturated with RPA for homologous pairing but does not appear to be more active than Rad51 when an RPA-free ssDNA template is used. Rad52 does not substitute for RPA in the pairing and strand exchange reaction nor does it lower the dependence of the reaction on Rad51 or RPA.

Highlights

IntroductionGenetic recombination is mediated by genes of the RAD52 epistasis group
In eukaryotic organisms, genetic recombination is mediated by genes of the RAD52 epistasis group
Effects of RPA and Rad52 on Rad51-mediated Strand Exchange—We examined the level of strand exchange reaction products (Fig. 1A) by fixing the amount of Rad51 (10 ␮M), and we varied the concentration of RPA (0.4 –2.8 ␮M), added either with Rad51 (Fig. 1B, panel I) or after Rad51 has already nucleated onto the ssDNA (Fig. 1B, panel II), as in the standard reaction

Summary

Introduction

Genetic recombination is mediated by genes of the RAD52 epistasis group. Studies on RecA, its bacteriophage T4 counterpart UvsX, and eukaryotic Rad have indicated that they mediate the homologous DNA pairing and strand exchange reaction that forms heteroduplex DNA during recombination. Rad52 [12, 13, 15] and the heterodimer of the Rad and Rad proteins [14] have been found to promote heteroduplex formation when there is a need for Rad to compete with RPA for binding sites on the ssDNA These ancillary protein factors, or mediators [15, 16], are functionally equivalent to the E. coli RecO-RecR complex [17] and T4 UvsY protein (18 –21), which allow their cognate recombinases RecA and UvsX to gain access to ssDNA already coated with the ssDNA binding factor. We describe biochemical studies that enable us to begin understanding the biochemical properties and the mediator function of Rad in greater detail

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