DNA DAMAGE REPAIR

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH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 中心，不一定是研究者的机构。 Ku 70/Ku 80异二聚体或Ku是双链断裂（DSB）修复的非同源末端连接（NHEJ）途径的中心组分。因为Ku形成了一个DSB穿过的环，它可能在修复过程中拓扑地附着在DNA上。其去除机制尚不清楚。使用一种方法来识别招募到非洲爪蟾卵提取物中的DSB的蛋白质，我们表明，含有DSB的DNA积累的Skp 1-Cul 1-F-盒复合物和K48连接的多泛素化蛋白的成员，除了已知的修复蛋白。我们证明，Ku 80是降解响应DSB在泛素介导的方式。引人注目的是，K48连接的多泛素化，而不是蛋白酶体降解，是从DNA中有效去除Ku 80所必需的。这种去除是DNA长度依赖性的，因为Ku 80保留在双链体寡核苷酸上。最后，NHEJ完成和从DNA中去除Ku 80是彼此独立的。我们建议，DSB诱导的Ku 80泛素化提供了一种机制，有效地消除Ku从DNA的前和后修复过程。描述这项工作的手稿已经出版： Ku 80通过双链断裂诱导的泛素化从DNA中去除。Postow L，Ghenoiu C，Woo EM，Krutchinsky AN，Chait BT，Funabiki H. J Cell Biol.2008 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.