Mechanism of DNA replication initiation in Saccharomyces cerevisiae

总结 DNA复制应激已被认为是基因组不稳定性的主要来源， 包括癌症在内的多种疾病因此，了解复制的分子基础 压力，以及相反，如何准确，完整和快速的染色体DNA复制是在 正常的细胞增殖，对于理解维持或威胁基因组的机制至关重要 正常发育和疾病期间的稳定性。在这里，我们提出的实验， 阐明了真核DNA复制机制的内在机制及其对 不同的复制应激条件。在上一个赠款周期，我们已经产生了一个完全重组 基于纯化的芽殖酵母蛋白质的起始依赖性DNA复制系统。该系统构成了 下一个资助周期我们实验室研究的中心平台。在目标1中，我们将利用独特的 该系统的生物化学易处理性以阐明真核复制DNA解旋酶的机制， CMG（Cdc 45-Mcm 2 -7-GINS），其位于复制体的中心。CMG在复制性 来自生命的三个领域的DNA解旋酶，因为其解旋酶马达，Mcm 2 -7复合物， 6个不同的亚基。造成这种复杂性的原因仍然不清楚。有趣的是，我们发现了一个 以前未认识到的重要作用，独特的非结构化N-末端尾部的Mcm 2。我们的初步 数据表明该结构域参与复制体的多种功能，包括DNA解旋， 引发和染色质复制。我们建议描述这种意想不到的功能多功能性， 解旋酶结构域作为阐明真核复制体内在机制的门户。的 目标2和目标3的重点将是扩展基本的DNA复制系统，用于DNA复制的研究 应力机制Aim 2的实验建立在我们最初的单向复制体重建的基础上 与R环碰撞，R环是一种共转录形成的核酸结构， 对所有生物体基因组稳定性的主要威胁。我们发现R环形成了一个内在的屏障， 复制体进程通过创建一个物理块到分叉。大量的蛋白质，如 解旋酶和RNase H-型核酸酶，已经涉及通过以下方式促进体内DNA复制： 解析R环然而，其机制在很大程度上是未知的。我们已经净化了这些附属品 酶，并将表征它们在促进通过R环的叉进展中的作用。目标3的基础 是我们的观察，dNTP耗竭，复制应激的一种最受研究的形式， 重组系统中复制体进程与DNA合成的解偶联。我们利用这种反应 研究检查点调节停滞的分叉稳定性的机制，重点是中央 检查点效应激酶，Rad 53。

Summary DNA replication stress has been recognized as a major source for the genome instability associated with numerous diseases, including cancer. Therefore, understanding the molecular basis of replication stress and, conversely, how accurate, complete, and rapid chromosomal DNA replication is achieved during normal cell proliferation, is crucial for understanding the mechanisms that maintain or threaten genome stability during normal development and disease, respectively. Here we propose experiments that will illuminate both the intrinsic mechanism of the eukaryotic DNA replication machinery and its response to diverse replication stress conditions. In the previous grant cycle we have generated a fully reconstituted origin-dependent DNA replication system based on purified budding yeast proteins. This system forms the central platform for research in our lab over the next funding cycle. In Aim 1 we will exploit the unique biochemical tractability of this system to elucidate the mechanism of the eukaryotic replicative DNA helicase, CMG (Cdc45-Mcm2-7-GINS), which is at the center of the replisome. The CMG is unique among replicative DNA helicases from the three domains of life in that its helicase motor, the Mcm2-7 complex, is composed of 6 distinct subunits. The reasons for this complexity are still obscure. Intriguingly, we have identified a previously unrecognized essential role for the unique unstructured N-terminal tail of Mcm2. Our preliminary data indicate that this domain is involved in multiple functions at the replisome, including DNA unwinding, priming, and chromatin replication. We propose to characterize the unexpected functional versatility of this helicase domain as a gateway to the elucidation of the intrinsic mechanism of a eukaryotic replisome. The focus of Aims 2 and 3 will be to expand the basic DNA replication system for the study of DNA replication stress mechanisms. Experiments in Aim2 build on our original reconstitution of unidirectional replisome collisions with R-loops, a co-transcriptionally formed nucleic acid structure that has been recognized to pose a major threat to genome stability in all organisms. We find that R-loops form an intrinsic barrier to replisome progression by creating a physical block to the fork. A large number of proteins, such as helicases and RNase H-type nucleases, have been implicated in promoting DNA replication in vivo by resolving R-loops. However, the mechanisms are largely unknown. We have purified these accessory enzymes and will characterize their role in promoting fork progression through R-loops. The basis for Aim 3 is our observation that dNTP depletion, one of the most-studied forms of replication stress, causes uncoupling of replisome progression from DNA synthesis in the reconstituted system. We use this reaction to investigate the mechanism by which the checkpoint regulates stalled fork stability, focusing on the central checkpoint effector kinase, Rad53.