Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.
Department of Biochemistry & Molecular Biophysics, Columbia University, New York, NY 10032, USA.
Mol Cell. 2019 Dec 5;76(5):699-711.e6. doi: 10.1016/j.molcel.2019.08.017. Epub 2019 Sep 18.
Rad52 is a key factor for homologous recombination (HR) in yeast. Rad52 helps assemble Rad51-ssDNA nucleoprotein filaments that catalyze DNA strand exchange, and it mediates single-strand DNA annealing. We find that Rad52 has an even earlier function in HR in restricting DNA double-stranded break ends resection that generates 3' single-stranded DNA (ssDNA) tails. In fission yeast, Exo1 is the primary resection nuclease, with the helicase Rqh1 playing a minor role. We demonstrate that the choice of two extensive resection pathways is regulated by Rad52. In rad52 cells, the resection rate increases from ∼3-5 kb/h up to ∼10-20 kb/h in an Rqh1-dependent manner, while Exo1 becomes dispensable. Budding yeast Rad52 similarly inhibits Sgs1-dependent resection. Single-molecule analysis with purified budding yeast proteins shows that Rad52 competes with Sgs1 for DNA end binding and inhibits Sgs1 translocation along DNA. These results identify a role for Rad52 in limiting ssDNA generated by end resection.
Rad52 是酵母中同源重组（HR）的关键因素。Rad52 有助于组装 Rad51-ssDNA 核蛋白丝，催化 DNA 链交换，并介导单链 DNA 退火。我们发现 Rad52 在 HR 中具有更早的功能，它限制了 DNA 双链断裂末端的切除，从而产生 3'单链 DNA（ssDNA）尾巴。在裂殖酵母中，Exo1 是主要的切除核酸酶，解旋酶 Rqh1 起次要作用。我们证明了两种广泛的切除途径的选择由 Rad52 调节。在 rad52 细胞中，切除率从约 3-5 kb/h 增加到约 10-20 kb/h，这是一种依赖于 Rqh1 的方式，而 Exo1 变得可有可无。芽殖酵母 Rad52 也抑制 Sgs1 依赖性切除。用纯化的芽殖酵母蛋白进行的单分子分析表明，Rad52 与 Sgs1 竞争 DNA 末端结合，并抑制 Sgs1 在 DNA 上的易位。这些结果确定了 Rad52 在限制末端切除产生的 ssDNA 方面的作用。
