Replication resolved in the living cell: Replisome dynamics, stability and structure.

摘要及时和忠实的复制对所有生命系统中的细胞增殖至关重要。复制 功能障碍导致突变，DNA断裂和细胞死亡，所有这些都在人类疾病中起着核心作用。 包括癌症在内的疾病和耐药性细菌病原体的发展。最近，我们 实验室发现，复制和转录机制之间的冲突增加了， 突变率以及在复制过程中引起复制体复合物的显著不稳定性。 我们的长期目标是剖析机制，细胞反应和生物和医学 转录复制冲突的后果。这项补助金的目的是描述 和稳定性的复制体，特别是在响应转录诱导的冲突， 单分子灵敏度。根据我们最近的观察，这一提议的中心假设是 复制体结构是复制的关键调节器和避免冲突的机制。 我们的理由是，我们将获得对复制过程以及复制的基本见解 通过研究活细胞中的复制体结构，一次一个与单分子敏感性相冲突。 我们的具体目标是联合收割机结构和功能分析相结合：目标1描述了一个程序，以表征 高分辨率下复制体的动态组织。目的2描述了一个功能分析的 复制体崩溃后的交换、重启和恢复机制。目标3描述了 假设细胞组织是减少复制冲突的关键机制。我们的前期工作 已经改变了对复制过程的基本理解， 不连续的拟议的工作是重要的，因为它将继续这一计划，探索基本的， 但关于复制体结构和冲突的细胞机制的未经检验的假设 解决和避免。这项研究是创新的，因为我们应用了一个跨学科的 研究活细胞中具有单分子敏感性的复制体的方法。目前还没有其他实验 在体内用这种分辨率探测了复制体结构，因此这项工作具有很大的潜力来揭示 对复制体结构和功能的新的和基本的见解。

Summary. Timely and faithful replication is essential to cellular proliferation in all living systems. Replication malfunction leads to mutations, breaks in the DNA, and cell death, all of which play central roles in human diseases including cancer and the development of antibiotic-resistant bacterial pathogens. Very recently, our laboratories have discovered that conflicts between the replication and transcription machineries increase mutation rates as well as cause significant instability of the replisome complex during the replication process. Our long-term goal is to dissect the mechanisms, cellular responses and biological and medical consequences of transcription-replication conflicts. The objective of this grant is to characterize the structure and stability of the replisome in general, and in particular, in response to transcription-induced conflicts with single-molecule sensitivity. Motivated by our recent observations, the central hypothesis of this proposal is that replisome structure is a critical regulator of replication and a mechanism of conflict avoidance. Our rationale is that we will gain fundamental insight into both the replication process as well as replication conflicts by studying the replisome structure in living cells one conflict at a time with single-molecule sensitivity. Our specific aims combine structural and functional analyses: Aim 1 describes a program to characterize the dynamic organization of the replisome at high-resolution. Aim 2 describes a functional analysis of the mechanisms for exchange, restart and recovery after replisome collapse. Aim 3 describes the test of the hypothesis that cellular organization is key mechanism for reducing replication conflicts. Our preliminary work has already changed the fundamental understanding of the replication process by demonstrating that it is discontinuous. The proposed work is significant since it will continue this program by probing fundamental, but untested assumptions about the structure of the replisome and the cellular mechanisms of conflict resolution and avoidance. The proposed research is innovative because we apply an interdisciplinary approach to study the replisome with single-molecule sensitivity in living cells. No other experiments have yet probed the replisome structure with this resolution in vivo and therefore the work has great potential to reveal both novel and fundamental insights into replisome structure and function.