Khade Nilesh V, Sugiyama Tomohiko
Department of Biological Sciences, Ohio University, Athens, Ohio, United States of America.
PLoS One. 2016 Jun 30;11(6):e0158436. doi: 10.1371/journal.pone.0158436. eCollection 2016.
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.
酵母Rad52（yRad52）在同源DNA重组（HR）中具有两个重要功能：使互补单链DNA（ssDNA）分子退火，并将Rad51重组酶招募到ssDNA上（重组介导活性）。其人类同源物（hRAD52）在HR中的作用较小，且明显缺乏介导活性。在此我们表明，yRad52能够在体外将人类Rad51（hRAD51）加载到与酵母RPA复合的ssDNA上。这在生化上等同于介导活性，因为它依赖于yRad52的C端Rad51结合区域以及功能性的Rad52-RPA相互作用。据报道，yRad52和hRAD52的N端三分之二对于结合和使ssDNA退火至关重要。尽管在yRad52的C端区域发现了第二个DNA结合区域，但其在ssDNA退火中的作用尚不清楚。在本文中，我们还表明yRad52的C端区域而非hRAD52的C端区域参与ssDNA退火。这表明第二个DNA结合位点是yRad52高效进行ssDNA退火所必需的。我们提出了一个更新的Rad52介导的ssDNA退火模型。
