End resection in homologous recombination (HR) and HR-mediated repair of DNA double-strand breaks (DSBs) removes several kilobases from 5′ strands of DSBs, but 3′ strands are exempted from degradation. The mechanism by which the 3′ overhangs are protected has not been determined. Here, we established that the protection of 3′ overhangs is achieved through the transient formation of RNA-DNA hybrids. The DNA strand in the hybrids is the 3′ ssDNA overhang, while the RNA strand is newly synthesized. RNA polymerase III (RNAPIII) is responsible for synthesizing the RNA strand. Furthermore, RNAPIII is actively recruited to DSBs by the MRN complex. CtIP and MRN nuclease activity is required for initiating the RNAPIII-mediated RNA synthesis at DSBs. A reduced level of RNAPIII suppressed HR, and genetic loss > 30 bp increased at DSBs. Thus, RNAPIII is an essential HR factor, and the RNA-DNA hybrid is an essential repair intermediate for protecting the 3′ overhangs in DSB repair.

同源重组 (HR) 中的末端切除和 HR 介导的 DNA 双链断裂 (DSB) 修复从 DSB 的 5' 链中去除了几千个碱基，但 3' 链免于降解。尚未确定保护 3' 突出端的机制。在这里，我们确定 3' 突出端的保护是通过 RNA-DNA 杂交体的瞬时形成来实现的。杂交体中的 DNA 链是 3' ssDNA 突出端，而 RNA 链是新合成的。RNA 聚合酶 III (RNAPIII) 负责合成 RNA 链。此外，RNAPIII 被 MRN 复合体积极招募到 DSB。在 DSB 上启动 RNAPIII 介导的 RNA 合成需要 CtIP 和 MRN 核酸酶活性。RNAPIII 水平的降低抑制了 HR，并且在 DSB 处遗传丢失 > 30 bp 增加。因此，