Mechanisms of DNA Replication, Chromosome Compaction, and Chromosome Unlinking

总结 遗传信息的准确传递需要染色体DNA的完全复制， 细胞分裂周期复制分叉的有序进展受到模板的挑战 损伤，缓慢移动和停滞的RNA聚合酶，以及冻结的DNA-蛋白质复合物，使叉子停止。 失速叉是基因组不稳定的焦点，导致遗传改变，并可能导致癌症。停滞 叉必须被重塑/修复并且复制重新开始/继续以维持基因组稳定性。 我们已经开发了一种大肠杆菌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 复制体在体外停滞的所有方面。在本提案中，我们调查了应对以下问题的综合网络： 细菌用来保持基因组完整性的DNA损伤。我们问：（i）停滞的分叉如何贡献 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之间的交换动力学是什么 聚合酶IV和DNA聚合酶III在复制体介导的跨病变旁路？（四）如何 复制体克服了与RNA聚合酶的碰撞，RNA聚合酶本身由于DNA模板损伤而停滞。 我们将开始应用我们的专业知识，以解决这些问题，使用人类复制蛋白质，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、 和细胞分裂时染色体分离。未能实现适当的染色体组织， 分离会导致DNA断裂，导致遗传物质在下一个群体中的不均匀分布。 一代染色体组织包括两种主要机制：拓扑维持和 蛋白质介导的DNA包装。前者通过调节网络的拓扑结构来防止纠缠。 DNA，解决不需要的链烷和结。后者塑造染色体的构象， 提高任何特定大分子交易的效率。我们的分析集中在相互作用 我们发现的细胞condesin，MukB和细胞decatenase拓扑异构酶IV之间的关系， 我们已经证明了这是染色体正确压实和分离所必需的。我们问：（一）什么是 MukB辅助蛋白MukE和MukF及MukB ATP水解在染色体中的作用 压缩？(ii)DNA-MukB-Topo IV结构的形成导致染色体 压缩？(iii)染色体致密化的缺陷如何影响DNA代谢过程， 修理？以及（iv）假定的细菌粘附素RecN如何在双链断裂修复中起作用， 子链间隙修复

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?