DNA DAMAGE REPAIR

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 Ku70/Ku80异源二聚体或Ku是双链断裂(DSB)修复的非同源末端连接(NHEJ)途径的中心成分。因为Ku形成一个环，DSB穿过它，它很可能在修复过程中以拓扑方式连接到DNA上。去除它的机制尚不清楚。使用一种方法来鉴定非洲爪哇卵提取物中招募到DSB的蛋白质，我们发现含有DSB的DNA除了积累已知的修复蛋白外，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。我们证明了Ku80以泛素介导的方式被DSB降解。值得注意的是，K48连接的多泛素化，而不是蛋白酶体的降解，是有效地从DNA中去除Ku80所必需的。这种去除依赖于DNA长度，因为Ku80保留在双链寡核苷酸上。最后，NHEJ的完成和从DNA中去除Ku80是相互独立的。我们认为，DSB诱导的Ku80泛素化为在配对前和配对后有效地从DNA中消除Ku提供了一种机制。描述这项工作的手稿已经出版： 通过双链断裂诱导泛素化从DNA中去除Ku80。L，吴恩美，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.