The mechanism of vertebrate DNA replication termination

 描述（由申请人提供）：脊椎动物染色体从数千个复制起点复制。在每个起点，两个复制叉建立在相反的方向，复制DNA，因为他们去。当聚合分叉相遇时，复制终止。终止包括DNA合成的局部完成、子分子的解链和复制体的解体。不能正确执行这些步骤会导致基因组不稳定，这是大多数癌症的标志。尽管它的重要性，终止的机制是知之甚少，主要是因为终止事件的时间和位置没有很好地定义。因此，终止是很难研究使用常规的方法，如染色质免疫沉淀。为了克服这些挑战，我们开发了一种方法来诱导本地化和同步终止事件在非洲爪蟾卵提取物。将含有乳糖操纵子（lacO）位点阵列的质粒与乳糖阻遏子（LacR）一起孵育，并加入到鸡蛋提取物中。DNA复制起始于质粒上的某个地方，两个分叉会聚在LacR阵列的外边缘，在那里它们停止。在添加IPTG后，LacR解离，叉立即恢复伸长，并且它们在lacO阵列内的小区域中同步地会聚和终止。使用这种方法，我们已经进行了最详细的机械解剖终止日期，导致一个全新的模型，这一过程。我们发现，有没有检测到的DNA合成的放缓叉收敛和解离的两个复制CMG解旋酶发生后，领导链连接到下游冈崎片段的收敛叉。这一观察结果表明，当CMG到达下游冈崎片段时，它们从ssDNA移动到dsDNA上，从那里它们被卸载。我们还发现，CMG卸载涉及其MCM7亚基的泛素化和p97 ATP酶的作用。在这个提议中，我们将使用我们新的无细胞系统来进一步阐明复制终止的机制。我们将测试我们的新假设，即CMG与dsDNA的相互作用触发CMG卸载（目的1）。我们将鉴定使MCM7泛素化以促进CMG卸载的E3泛素连接酶，以及与p97合作以从DNA中提取CMG的任何衔接蛋白（Aim 2）。我们还将确定E3泛素连接酶和p97如何识别终止的CMG。最后，我们将讨论CMG卸载失败如何影响DNA复制和DNA修复（目标3）。我们的实验将导致第一次全面的真核生物复制终止的分子描述，并阐明如何适当调节这一过程抑制基因组的不稳定性。

 DESCRIPTION (provided by applicant): Vertebrate chromosomes are replicated from thousands of origins of replication. At each origin, two replication forks are established that travel in opposite directions, copying DNA as they go. When converging forks meet, replication terminates. Termination involves local completion of DNA synthesis, decatenation of daughter molecules, and replisome disassembly. Failure to properly execute these steps leads to genome instability, a hallmark of most cancers. Despite its importance, the mechanism of termination is poorly understood, largely because the timing and location of termination events is not well-defined. As a result, termination is difficult to study using conventional approaches such as chromatin immunoprecipitation. To overcome these challenges, we developed an approach to induce localized and synchronous termination events in Xenopus egg extracts. A plasmid containing an array of lac operator (lacO) sites is incubated with lac repressor (LacR) and added to egg extract. DNA replication initiates somewhere on the plasmid and two forks converge on the outer edges of the LacR array, where they stall. Upon addition of IPTG, LacR dissociates, forks immediately resume elongation, and they converge and terminate synchronously in a small region within the lacO array. Using this approach, we have undertaken the most detailed mechanistic dissection of termination to date, leading to a fundamentally new model of this process. We show that there is no detectable slowing of DNA synthesis as forks converge and that dissociation of the two replicative CMG helicases occurs after leading strands are ligated to downstream Okazaki fragments of the converging fork. This observation implies that when CMGs reach the downstream Okazaki fragment, they move from ssDNA onto dsDNA, from where they are unloaded. We also found that CMG unloading involves ubiquitylation of its MCM7 subunit and the action of the p97 ATPase. In this proposal, we will use our new cell-free system to further elucidate the mechanism of replication termination. We will test our novel hypothesis that the interaction of CMG with dsDNA triggers CMG unloading (Aim 1). We will identify the E3 ubiquitin ligase that ubiquitylates MCM7 to promote CMG unloading, as well as any adaptor proteins that cooperate with p97 to extract CMG from DNA (Aim 2). We will also determine how the E3 ubiquitin ligase and p97 recognize terminated CMGs. Finally, we will address how failure to unload CMG affects DNA replication and DNA repair (Aim 3). Our experiments will lead to the first comprehensive molecular description of eukaryotic replication termination and elucidate how the proper regulation of this process suppresses genome instability.