Mechanism of DNA replication initiation in Saccharomyces cerevisiae

摘要 DNA复制应激已被认为是基因组不稳定性的主要来源 患有多种疾病，包括癌症。因此，了解复制的分子基础 压力，反之，如何准确、完整和快速地实现染色体DNA复制 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 在正常发育和疾病期间分别保持稳定。在这里，我们提出的实验将 阐明真核DNA复制机制的内在机制及其对 不同的复制胁迫条件。在上一个赠款周期中，我们生成了一个完全重组的 基于纯芽酵母蛋白的来源依赖的DNA复制系统。这一系统构成了 我们实验室在下一个资助周期中的研究中心平台。在目标1中，我们将利用独特的 该体系的生化可操作性为阐明真核复制DNA解旋酶的作用机制奠定了基础 CMG(cdc45-mcm2-7-gins)，位于复制体的中心。CMG在复制中是独一无二的 Dna解旋酶来自生命的三个领域，因为它的解旋酶马达mcm2-7复合体由 由6个不同的亚基组成。这种复杂性的原因仍然不清楚。有趣的是，我们发现了一个 之前未被认识的独特的非结构化的McM2的N端尾巴的重要作用。我们的预赛 数据表明，该结构域参与了复制体的多种功能，包括DNA解离， 启动和染色质复制。我们建议将其出人意料的功能多功能性描述为 解旋酶结构域作为阐明真核复制体内在机制的门户。这个 AIMS 2和AIMS 3的重点将是为研究DNA复制扩展基本DNA复制系统 压力机制。AIM2的实验建立在我们最初的单向复制体重构体基础上 与R环的碰撞，R环是一种共转录形成的核酸结构，已被认为构成 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障碍 通过在叉子上创建一个物理块来复制进程。大量的蛋白质，如 解旋酶和核糖核酸酶H型核酸酶通过以下途径促进体内DNA复制 解决R-环路。然而，机制在很大程度上是未知的。我们已经提纯了这些配件 并将表征它们在通过R-环促进分叉进展中的作用。目标3的基础 我们的观察是dNTP耗尽，一种被研究最多的复制应激形式，导致 重组系统中复制体进程与DNA合成的解偶联。我们使用这种反应 研究检查点调节停滞的叉子稳定性的机制，重点放在中央 检查点效应蛋白激酶，RAD53。

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.