Mechanisms of DNA Replication, Chromosome Compaction, and Chromosome Unlinking

DNA 复制、染色体压缩和染色体解联机制

基本信息

批准号：
10373984
负责人：
KENNETH J MARIANS
金额：
$ 102.86万
依托单位：
SLOAN-KETTERING INST CAN RESEARCH
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：
2018-04-01 至 2023-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10373984
关键词：
ATP Hydrolysis Address Affect Bacteria Bypass Catenanes Cell Cycle Cell division Cells Chromosomal Duplication Chromosome Segregation Chromosome Structures Chromosomes DNA DNA Damage DNA Packaging DNA Polymerase III DNA Polymerase beta DNA Repair DNA Topoisomerase IV DNA biosynthesis DNA replication fork DNA-Directed RNA Polymerase Daughter Defect Double Strand Break Repair Escherichia coli Failure Freezing Genetic Materials Genetic Transcription Genome Stability Genomic Instability Human In Vitro Lead Lesion Maintenance Malignant Neoplasms Mediating Metabolism Modeling Molecular Conformation Mutation Pathway interactions Proteins Role SOS Response Shapes Structure System Time Topoisomerase Transact cohesin condensin daughter cell genetic information genome integrity next generation preservation prevent protein complex repaired response segregation single molecule transmission process

项目摘要

Summary Accurate transmission of the genetic information requires complete duplication of the chromosomal DNA each cell division cycle. The orderly progression of replication forks is challenged by encounters with template damage, slow moving and arrested RNA polymerases, and frozen DNA-protein complexes that stall the fork. Stalled forks are foci for genomic instability that causes genetic alterations and can give rise to cancer. Stalled forks must be remodeled/repaired and replication restarted/continued in order to maintain genomic stability. We have developed an Escherichia coli DNA replication system that allows us to analyze the consequences of collision of the replisome with leading-strand template damage and with which we can model all aspects of replisome stalling in vitro. In this proposal we investigate the integrated network of responses to DNA damage that the bacterium uses to preserve genomic integrity. We ask: (i) how do stalled forks contribute to induction of the DNA damage (SOS) response? (ii) What is the mechanism of the UmuDC DNA replication checkpoint elaborated by the SOS response? (iii) What are the dynamics of exchange between DNA polymerase IV and DNA polymerase III during replisome-mediated trans-lesion bypass? And (iv), how do replisomes overcome collisions with RNA polymerases that are themselves stalled by DNA template damage. We will begin to apply our expertise to address these questions using human replication proteins and are also expanding our analyses by using single molecule approaches. Coordinating the structural organization of chromosomes is essential for DNA replication, transcription, and chromosome segregation during cell division. Failure to achieve proper chromosomal organization during separation can result in DNA breakage, leading to an uneven distribution of the genetic material to the next generation. Chromosomal organization involves two principal mechanisms: topological maintenance and protein-mediated packaging of the DNA. The former prevents entanglement by regulating the topology of the DNA, resolving unwanted catenanes and knots. The latter shapes the conformation of chromosomes, increasing the efficiency of any particular macromolecular transaction. Our analyses focus on the interaction between the cellular condesin, MukB, and the cellular decatenase topoisomerase IV that we discovered and that we have shown to be required for proper chromosome compaction and segregation. We ask: (i) what is the role of the MukB accessory proteins MukE and MukF and MukB ATP hydrolysis in chromosome compaction? (ii) What are the DNA-MukB-Topo IV structures that are formed that lead to chromosome compaction? (iii) How do defects in chromosome compaction affect DNA metabolic processes such as DNA repair? And (iv) how does the presumptive bacterial cohesin, RecN, function in double-strand break repair and daughter-strand gap repair?

概括遗传信息的准确传递需要染色体DNA的完整复制细胞分裂周期。复制叉的有序进展受到模板遭遇的挑战损伤、缓慢移动和停滞的 RNA 聚合酶以及使叉子停滞的冷冻 DNA-蛋白质复合物。停滞的叉子是基因组不稳定的焦点，会导致基因改变并可能引发癌症。停滞不前为了维持基因组的稳定性，必须重塑/修复分叉并重新启动/继续复制。我们开发了大肠杆菌 DNA 复制系统，使我们能够分析复制体与前导链模板损伤碰撞的后果，我们可以用它来建模体外复制体停滞的所有方面。在本提案中，我们研究了响应的综合网络细菌利用 DNA 损伤来保持基因组完整性。我们问：（i）停滞的叉子有何贡献诱导 DNA 损伤 (SOS) 反应？ (ii) UmuDC DNA复制的机制是什么 SOS 响应详细说明的检查点？ (iii) DNA 之间的交换动态是怎样的复制体介导的跨损伤旁路期间的聚合酶 IV 和 DNA 聚合酶 III？以及 (iv)，如何做复制体克服了与 RNA 聚合酶的碰撞，而 RNA 聚合酶本身因 DNA 模板损伤而停滞。我们将开始运用我们的专业知识，利用人类复制蛋白来解决这些问题，并且通过使用单分子方法扩展我们的分析。协调染色体的结构组织对于 DNA 复制、转录、以及细胞分裂过程中的染色体分离。未能实现适当的染色体组织分离会导致 DNA 断裂，从而导致遗传物质不均匀地分布到下一个区域。一代。染色体组织涉及两个主要机制：拓扑维持和蛋白质介导的 DNA 包装。前者通过调节拓扑结构来防止纠缠 DNA，解决不需要的索链和结。后者塑造染色体的构象，提高任何特定大分子交易的效率。我们的分析重点是交互细胞压缩蛋白 MukB 和我们发现的细胞十链酶拓扑异构酶 IV 之间我们已经证明这是适当的染色体压缩和分离所必需的。我们问：（i）什么是 MukB 辅助蛋白 MukE 和 MukF 以及 MukB ATP 水解在染色体中的作用压实？ (ii) 形成的导致染色体的 DNA-MukB-Topo IV 结构是什么？压实？ (iii)染色体压缩缺陷如何影响DNA等DNA代谢过程维修？ (iv) 假定的细菌粘连蛋白 RecN 在双链断裂修复中如何发挥作用？子链间隙修复？