Department of Molecular Genetics, University of Toronto, Toronto, Canada.
Mol Cell Biol. 2014 Jan;34(1):132-45. doi: 10.1128/MCB.00639-13. Epub 2013 Nov 4.
The minichromosome maintenance (MCM) complex, which plays multiple important roles in DNA replication, is loaded onto chromatin following mitosis, remains on chromatin until the completion of DNA synthesis, and then is unloaded by a poorly defined mechanism that involves the MCM binding protein (MCM-BP). Here we show that MCM-BP directly interacts with the ubiquitin-specific protease USP7, that this interaction occurs predominantly on chromatin, and that MCM-BP can tether USP7 to MCM proteins. Detailed biochemical and structure analyses of the USP7-MCM-BP interaction showed that the (155)PSTS(158) MCM-BP sequence mediates critical interactions with the TRAF domain binding pocket of USP7. Analysis of the effects of USP7 knockout on DNA replication revealed that lack of USP7 results in slowed progression through late S phase without globally affecting the fork rate or origin usage. Lack of USP7 also resulted in increased levels of MCM proteins on chromatin, and investigation of the cause of this increase revealed a defect in the dissociation of MCM proteins from chromatin in mid- to late S phase. This role of USP7 mirrors the previously described role for MCM-BP in MCM complex unloading and suggests that USP7 works with MCM-BP to unload MCM complexes from chromatin at the end of S phase.
微染色体维持 (MCM) 复合物在 DNA 复制中发挥多种重要作用，在有丝分裂后加载到染色质上，在 DNA 合成完成之前一直保留在染色质上，然后通过一个定义不明确的机制卸载，该机制涉及 MCM 结合蛋白 (MCM-BP)。在这里，我们表明 MCM-BP 直接与泛素特异性蛋白酶 USP7 相互作用，这种相互作用主要发生在染色质上，并且 MCM-BP 可以将 USP7 固定在 MCM 蛋白上。对 USP7-MCM-BP 相互作用的详细生化和结构分析表明，(155)PSTS(158)MCM-BP 序列介导与 USP7 的 TRAF 结构域结合口袋的关键相互作用。分析 USP7 敲除对 DNA 复制的影响表明，缺乏 USP7 会导致晚期 S 期进展缓慢，而不会全局影响叉速率或起始使用。缺乏 USP7 还导致染色质上 MCM 蛋白水平升高，并且对这种增加的原因的研究表明，在中晚期 S 期，MCM 蛋白从染色质上的解离存在缺陷。USP7 的这种作用反映了先前描述的 MCM-BP 在 MCM 复合物卸载中的作用，并表明 USP7 与 MCM-BP 一起在 S 期结束时从染色质上卸载 MCM 复合物。
