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

• 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
• 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

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 遗传信息的准确传输需要完全重复染色体DNA 细胞分裂周期。与模板的相遇挑战复制叉的有序进度 损坏，移动缓慢和捕捉的RNA聚合酶以及降低叉子的冷冻DNA蛋白复合物。 停滞的叉子是引起遗传改变并可能引起癌症的基因组不稳定性的焦点。停滞不前 必须对叉子进行重塑/修复并进行复制重新启动/续，以维持基因组稳定性。 我们已经开发了一个大肠杆菌DNA复制系统，使我们能够分析 与前链模板损伤相撞的后果，我们可以通过其建模 在体外停滞不前的所有方面。在此提案中，我们调查了综合响应网络 细菌用来保留基因组完整性的DNA损伤。我们问：（i）停滞的叉子如何贡献 诱导DNA损伤（SOS）反应？ （ii）UMUDC DNA复制的机制是什么 检查点由SOS响应详细说明？ （iii）DNA之间的交换动态是什么 聚合酶IV和DNA聚合酶III在重新介导的跨性质旁路期间？ （iv），如何 重新分裂克服了与DNA模板损伤阻滞的RNA聚合酶的碰撞。 我们将开始利用人类复制蛋白来应用我们的专业知识来解决这些问题，也是 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，至关重要 和细胞分裂期间的染色体分离。在期间未能实现适当的染色体组织 分离会导致DNA断裂，从而导致遗传物质分布不平均 一代。染色体组织涉及两种主要机制：拓扑维护和 蛋白质介导的DNA包装。前者通过调节 DNA，解决不需要的蛋白菜和结。后者塑造了染色体的构象， 提高任何特定的大分子交易的效率。我们的分析专注于互动 在我们发现的细胞凝结，MUKB和细胞脱发酶拓扑酶IV之间 我们已经证明我们需要适当的染色体压实和隔离。我们问：（i）什么是 MUKB附件蛋白Muke和Mukf和Mukb ATP水解在染色体中的作用 压实？ （ii）形成导致染色体的DNA-MUKB-TOPO IV结构 压实？ （iii）染色体压实的缺陷如何影响DNA代谢过程，例如DNA 维修？ （iv）假定细菌粘着素，recn，在双链断裂修复和 女儿链差距维修？