Roles of C-Terminal Region of Yeast and Human Rad52 in Rad51-Nucleoprotein Filament Formation and ssDNA Annealing

Nilesh V Khade,Tomohiko Sugiyama,Jennifer C Fung

doi:10.1371/journal.pone.0158436

Abstract

Yeast Rad52 (yRad52) has two important functions at homologous DNA recombination (HR); annealing complementary single-strand DNA (ssDNA) molecules and recruiting Rad51 recombinase onto ssDNA (recombination mediator activity). Its human homolog (hRAD52) has a lesser role in HR, and apparently lacks mediator activity. Here we show that yRad52 can load human Rad51 (hRAD51) onto ssDNA complexed with yeast RPA in vitro. This is biochemically equivalent to mediator activity because it depends on the C-terminal Rad51-binding region of yRad52 and on functional Rad52-RPA interaction. It has been reported that the N-terminal two thirds of both yRad52 and hRAD52 is essential for binding to and annealing ssDNA. Although a second DNA binding region has been found in the C-terminal region of yRad52, its role in ssDNA annealing is not clear. In this paper, we also show that the C-terminal region of yRad52, but not of hRAD52, is involved in ssDNA annealing. This suggests that the second DNA binding site is required for the efficient ssDNA annealing by yRad52. We propose an updated model of Rad52-mediated ssDNA annealing.

Highlights

Faithful repair of DNA double-strand breaks (DSBs) is crucial for genome stability
DNA strand exchange was stimulated by increasing amounts of Yeast Rad52 (yRad52) (Fig 1D and 1F)
Results presented in this paper (Figs 1 and 2) indicate that yRad52 can recruit human Rad51 (hRAD51) onto yRPA-single-stranded DNA (ssDNA) complex in vitro by a mechanism equivalent to the mediator activity

Summary

Introduction

Faithful repair of DNA double-strand breaks (DSBs) is crucial for genome stability. An unrepaired DSB may cause cell death and erroneous DSB repair may cause cancer [1,2,3]. Rad is one of the key proteins involved in DSB repair [4, 5]. The role of Rad in the DSB repair has been extensively studied in the yeast Saccharomyces cerevisiae, where Rad is required for DSB repair both in the high-fidelity homologous recombination (HR) pathway [6,7,8,9] and in an error-prone single strand annealing (SSA) pathway [10]. In vitro studies have shown that Rad can associate with single-stranded DNA (ssDNA) that is complexed with the ssDNA binding protein RPA [11, 12], and mediates two reactions that are crucial for HR and SSA pathways. The resulting Rad51-ssDNA nucleoprotein filament catalyzes DNA strand invasion, a key step of HR pathway [15]. Rad can anneal complementary ssDNA molecules in the SSA pathway [16]

Methods

Results

Conclusion