DNA DAMAGE REPAIR

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 Ku70/Ku80 异二聚体 (Ku) 是双链断裂 (DSB) 修复非同源末端连接 (NHEJ) 途径的核心组成部分。因为 Ku 形成了一个 DSB 穿过的环，所以它很可能在修复过程中以拓扑方式附着在 DNA 上。其去除机制尚不清楚。使用一种方法来鉴定非洲爪蟾卵提取物中 DSB 中招募的蛋白质，我们发现，除了已知的修复蛋白之外，含有 DSB 的 DNA 还积累了 Skp1-Cul1-F-box 复合体的成员和 K48 连接的多泛素化蛋白。我们证明 Ku80 以泛素介导的方式响应 DSB 而被降解。引人注目的是，从 DNA 中有效去除 Ku80 需要 K48 连接的多泛素化，而不是蛋白酶体降解。这种去除取决于 DNA 长度，因为 Ku80 保留在双链寡核苷酸上。最后，NHEJ 的完成和 Ku80 从 DNA 中的去除是相互独立的。我们认为 DSB 诱导的 Ku80 泛素化提供了一种在修复前和修复后过程中有效从 DNA 中消除 Ku 的机制。描述这项工作的手稿已经出版： 通过双链断裂诱导的泛素化从 DNA 中去除 Ku80。 Postow L、Ghenoiu C、Woo EM、Krutchinsky AN、Chait BT、Funabiki H. J 细胞生物学。 2008年11；182(3)：467-79。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The Ku70/Ku80 heterodimer, or Ku, is the central component of the nonhomologous end joining (NHEJ) pathway of double strand break (DSB) repair. Because Ku forms a ring through which the DSB threads, it likely becomes topologically attached to DNA during repair. The mechanism for its removal was unknown. Using a method to identify proteins recruited to DSBs in Xenopus laevis egg extract, we show that DSB-containing DNAs accumulate members of the Skp1-Cul1-F-box complex and K48-linked polyubiquitylated proteins in addition to known repair proteins. We demonstrate that Ku80 is degraded in response to DSBs in a ubiquitin-mediated manner. Strikingly, K48-linked polyubiquitylation, but not proteasomal degradation, is required for the efficient removal of Ku80 from DNA. This removal is DNA length dependent, as Ku80 is retained on duplex oligonucleotides. Finally, NHEJ completion and removal of Ku80 from DNA are independent from one another. We propose that DSB-induced ubiquitylation of Ku80 provides a mechanism to efficiently eliminate Ku from DNA for pre- and postrepair processes. A manuscript describing this work has been published: Ku80 removal from DNA through double strand break-induced ubiquitylation. Postow L, Ghenoiu C, Woo EM, Krutchinsky AN, Chait BT, Funabiki H. J Cell Biol. 2008 11;182(3):467-79.