The mechanism of vertebrate DNA replication termination

 描述(申请人提供)：脊椎动物的染色体从数千个复制来源复制而来。在每个起始点，建立了两个复制分叉，它们向相反的方向移动，在前进的过程中复制DNA。当聚合分叉相遇时，复制将终止。终止包括DNA合成的局部完成、子体分子的去连接和复制体的拆解。未能正确执行这些步骤会导致基因组不稳定，这是大多数癌症的一个特征。尽管终止机制很重要，但人们对终止机制知之甚少，这主要是因为终止事件的时间和地点没有明确定义。因此，很难使用常规方法，如染色质免疫沉淀来研究终止。为了克服这些挑战，我们开发了一种方法来在非洲爪哇卵提取液中诱导局部和同步终止事件。含有一组Lac操纵子(LACO)的质粒与Lac抑制物(LACR)孵育，并添加到鸡蛋提取物中。DNA复制在质粒上的某个地方开始，两个叉子在LACR阵列的外缘会合，在那里它们停滞。添加IPTG后，LACR解离，叉子立即恢复伸长，它们在LACO阵列内的一个小区域内同步收敛和终止。使用这种方法，我们进行了迄今为止最详细的终止机制剖析，导致了这一过程的根本新模式。我们表明，当分叉收敛时，没有可检测到的DNA合成放缓，并且在前导链连接到收敛分叉的下游Okazaki片段后，两个复制的CMG解旋酶发生解离。这一观察表明，当CMG到达下游的Okazaki片段时，它们从ssDNA转移到dsDNA，在那里它们被卸载。我们还发现CMG的卸载涉及其MCM7亚基的泛素化和p97 ATPase的作用。在这项提案中，我们将使用我们的新的无细胞系统来进一步阐明复制终止的机制。我们将检验我们的新假设，即CMG与dsDNA的相互作用触发CMG卸载(目标1)。我们将确定泛素化MCM7以促进CMG卸载的E3泛素连接酶，以及与p97合作从DNA中提取CMG的任何适配蛋白(目标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，我们将讨论未能卸载CMG如何影响DNA复制和DNA修复(目标3)。我们的实验将导致对真核细胞复制终止的第一个全面的分子描述，并阐明这一过程的适当调节如何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.