Mechanisms of DNA Replication, Chromosome Compaction, and Chromosome Unlinking

摘要 遗传信息的准确传递需要完全复制每条染色体的DNA 细胞分裂周期。复制分叉的有序发展受到模板遭遇的挑战 损坏，移动缓慢和停滞的RNA聚合酶，以及冻结的DNA-蛋白质复合体，使叉子失速。 失速的叉子是基因组不稳定的焦点，这种不稳定会导致基因变化，并可能导致癌症。陷入停滞 为了保持基因组的稳定性，必须对叉子进行改造/修复，并重新开始/继续复制。 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 复制体体外停滞的方方面面。在本提案中，我们调查了对以下各项的综合响应网络 细菌用来保持基因组完整性的DNA损伤。我们问：(I)失速的叉子有什么贡献 诱导DNA损伤(SOS)反应？(Ii)UmuDC DNA复制的机制是什么 SOS回应中详细说明的检查点？(Iii)DNA之间的交换动力学是什么 复制体介导跨病变搭桥术中的聚合酶IV和DNA聚合酶III？和(Iv)，如何 复制体克服了与RNA聚合酶的碰撞，而RNA聚合酶本身因DNA模板破坏而停滞不前。 我们将开始应用我们的专业知识来使用人类复制蛋白来解决这些问题，并正在 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。没有达到适当的染色体组织在 分离可能导致DNA断裂，导致遗传物质不均匀地分配给下一个 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。前者通过调节网络的拓扑结构防止纠缠 DNA，分解不需要的链环和绳结。后者塑造了染色体的构象， 提高任何特定大分子交易的效率。我们的分析重点放在互动上 在我们发现的细胞Condesin，MukB和细胞十烯酸酶拓扑异构酶IV之间，以及 我们已经证明，正确的染色体紧凑和分离是必需的。我们问：(I)什么是 MukB辅助蛋白MUKE和MUKF及MUKB ATP水解酶在染色体中的作用 压实？(Ii)形成染色体的DNA-MukB-Topo IV结构是什么 压实？(3)染色体紧凑缺陷如何影响DNA等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和修复中发挥作用 女儿股缝隙修复？

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?