Human DNA Replication Machines: Structure-function Studies

项目总结/摘要 基因组复制是生命的基本过程，几乎影响人类健康的各个方面。 因此，详细了解基因组复制机制对疾病的未来进展至关重要 诊断、药物设计和患者治疗。大部分人类DNA复制是由B家族完成的 DNA聚合酶Pol δ和Pol ε。然而，没有DNA引物，Pol δ和Pol ε不能开始合成。到 为了解决这个问题，一种称为引物酶的专门RNA聚合酶产生初始引物。然后 一种专门的B家族DNA聚合酶α（Pol α），与引发酶（primase）形成紧密的复合物（称为 primosome）用脱氧核糖核苷酸延伸RNA引物，然后将它们转换为Pol ε，开始 pol δ是冈崎病毒数百万个片段复制的起点， 落后的一段B家族的其余成员是DNA聚合酶β（Polymerase β，Polymerase β），它是DNA聚合酶β的关键参与者。 跨损伤DNA合成。目前对B家族DNA的理解仍然存在重大差距 聚合酶的功能，特别是关于密切协调聚合酶交易的关键因素， 复制叉。这种全球协调的关键组成部分是模板：引物机制 在前导链和滞后链的不对称合成期间从Pol α到Pol ε和Pol δ的切换， 计算Okazaki片段的长度，Pol δ和Pol ε向生产性伸长的转换。我们 发现Pol δ的辅助B亚基也与Pol δ的催化亚基形成复合物， 对于病变旁路过程中的聚合酶转换很重要。然而，这种转换的机制仍然存在， 未知解决这些挑战的最大障碍之一是结构信息不足， 特别是对于整个聚合酶复合物，包括Pol δ、Pol ε和Pol ε， 分子结构对于设计有意义的功能测定是必不可少的。基于我们之前 通过对原始体和Pol δ、Pol ε和Pol ε组分的生产性研究，我们提出了一个新的方向， 研究引物合成中紧密协调的事件，从Pol α到Pol ε的引物切换 和Pol δ，以及它们切换到精确伸长模式。对于拟议的研究，我们将应用X射线 晶体学和各种结构指导的生物化学和单分子实验。大多数这些 将使用体外重构的人复制体进行研究。

Project Summary/Abstract The genome replication is fundamental process of life that impacts virtually every aspect of human health. Therefore, a detailed understanding of genome replication mechanisms is vital for future advances in disease diagnosis, drug design, and patient treatment. The bulk of human DNA replication is performed by the B-family DNA polymerases Polδ and Polε. However, Polδ and Polε cannot begin synthesis without DNA primers. To circumvent this problem, a specialized RNA polymerase called primase generates the initial primers. Then, a dedicated B-family DNA polymerase α (Polα) working in a tight complex with primase (referred to as a primosome) extends the RNA primers with deoxyribonucleotides, before switching them to Polε for the start of leading-strand replication and to Polδ for the start of replication of each of the millions of Okazaki fragments of the lagging strand. The remaining member of the B-family is DNA polymerase ζ (Polζ), which is a key player in translesion DNA synthesis. A significant gap remains in the current understanding of B-family DNA polymerases' function, especially regarding the key factors that tightly coordinate polymerase transactions at the replication fork. The crucial components of this global coordination are the mechanisms of template:primer handover from Polα to Polε and Polδ during asymmetrical synthesis of both the leading and lagging strands, counting the length of Okazaki fragments, and the switch of Polδ and Polε to productive elongation. We discovered that the accessory B-subunit of Polδ also makes a complex with the catalytic subunit of Polζ, which is important for the polymerase switch during lesion bypass. However, the mechanism of this switch remains unknown. One of the biggest impediments in resolving these challenges is insufficient structural information, especially for entire polymerase complexes, including Polδ, Polε, and Polζ, as adequate knowledge of molecular structure is essential for the design of meaningful functional assays. Based on our previous productive studies of primosome and the components of Polδ, Polε, and Polζ, we propose a new direction of investigation that examines the tightly coordinated events in primer synthesis, primer handoff from Polα to Polε and Polδ, and their switch to accurate elongation mode. For the proposed studies, we will apply X-ray crystallography and a variety of structure-guided biochemical and single-molecule experiments. Most of these studies will be conducted using the in vitro reconstituted human replisome.