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

活细胞中的复制已解决：复制体动力学、稳定性和结构。

基本信息

批准号：
10158530
负责人：
Houra Merrikh
金额：
$ 34.53万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2022-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10158530
关键词：
Antibiotic Resistance Bacteria Bacterial Antibiotic Resistance Bacterial Model Biochemical Biochemical Genetics Biochemistry Biological Biological Models Cancer Etiology Cell Cycle Cell Death Cell Nucleus Cell Proliferation Cell physiology Cells Complex Conflict (Psychology)DNA DNA Structure DNA biosynthesis Development Due Process Ensure Eukaryotic Cell Frequencies Genetic Genetic Transcription Goals Grant Human Image Laboratories Life Malignant Neoplasms Medical Membrane Methods Microscopy Modeling Mutation Organelles Organism Play Process Proteins Recovery Replication-Associated Process Reporting Research Resolution Role Structural Models Structure Testing Time Transcription Process Work conflict resolution emerging antibiotic resistance experimental study human disease imaging modality in vivo innovation insight interdisciplinary approach interdisciplinary collaboration molecular imaging novel pathogenic bacteria programs response single molecule

项目摘要

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.

总结。及时而忠实的复制对于所有生命系统中的细胞增殖都是至关重要的。复制故障导致突变、DNA断裂和细胞死亡，所有这些都在人类中发挥着核心作用包括癌症在内的疾病和抗药性细菌病原体的发展。最近，我们的实验室发现，复制和转录机制之间的冲突增加在复制过程中，突变率以及导致复制体复合体的显著不稳定。我们的长期目标是剖析其机制、细胞反应以及生物学和医学转录-复制冲突的后果。这笔赠款的目的是描述这一结构一般情况下复制体的稳定性，特别是对转录诱导的与单分子敏感性。根据我们最近的观察，这一提议的中心假设是这种复制体结构是复制的关键调节因素和避免冲突的机制。我们的基本原理是，我们将从根本上深入了解复制过程和复制通过研究活细胞中的复制体结构，一次一个冲突，具有单分子敏感性。我们的具体目标结合了结构和功能分析：目标1描述了一个程序，以表征高分辨率复制体的动态组织。目标2描述了一种功能分析复制体崩溃后的交换、重新启动和恢复机制。目标3描述了测试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，证明它是不连续的。拟议的工作意义重大，因为它将通过探索基础、但关于复制体的结构和冲突的细胞机制的未经检验的假设解决和回避。拟议的研究是创新的，因为我们应用了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用这一分辨率在体内探测复制体结构，因此这项工作具有很大的揭示潜力对复制体结构和功能的新颖和基本的见解。