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

Yumiko Kurokawa,Yasuto Murayama,Nami Haruta-Takahashi,Hiroshi Iwasaki,Itaru Urabe,James E Harber

doi:10.1371/journal.pbio.0060088

Yumiko Kurokawa, Yasuto Murayama + Show 4 more

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https://doi.org/10.1371/journal.pbio.0060088

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Journal: PLoS Biology	Publication Date: Apr 1, 2008
Citations: 78	License type: CC BY 4.0

Affiliation: Yokohama City University, Osaka University

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

Highlights

Homologous recombination (HR) generates genetic diversity by rearranging DNA sequences using homologous DNA information
A series of analyses suggested that the assembly pathway for Rad51 on single-stranded DNA (ssDNA) in vivo is spatiotemporally regulated by replication protein A (RPA) and other Rad52 epistasis group proteins, such as Rad52 and Rad55–57 [5,6]
It is thought that a recombinase binds to single-stranded DNA to form a nucleoprotein filament called the presynaptic filament, and that this higher order structure engages in a search for homologous DNA sequences

Summary

Introduction

Homologous recombination (HR) generates genetic diversity by rearranging DNA sequences using homologous DNA information It is an important mechanism for repairing DNA double-stranded breaks (DSBs) and restarting stalled DNA replication forks. The Rad recombinase belongs to the Rad group and plays a key role in HR: Rad forms nucleoprotein filaments with single-stranded DNA (ssDNA), referred to as presynaptic filaments, and promotes strand exchange with donor DNA in an ATP-dependent manner. A series of analyses suggested that the assembly pathway for Rad on ssDNA in vivo is spatiotemporally regulated by replication protein A (RPA) and other Rad epistasis group proteins, such as Rad and Rad55–57 [5,6]. Rad assists in loading Rad onto RPA-coated ssDNA and in assembling the Rad nucleoprotein filament.

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