Human DNA Replication Machines: Structure-function Studies

项目摘要/摘要 基因组复制是生命的基本过程，几乎影响到人类健康的方方面面。 因此，对基因组复制机制的详细了解对于疾病的未来发展至关重要。 诊断、药物设计和病人治疗。人类DNA的大部分复制是由B家族执行的 DNA聚合酶POLδ和POLε。然而，POLδ和POLε在没有DNA引物的情况下不能开始合成。至 为了绕过这个问题，一种名为Primase的特殊RNA聚合酶产生了最初的引物。然后，一个 在与底物酶紧密的复合体中工作的专用B家族DNA聚合酶α(POLα Primosome)用脱氧核糖核苷酸延伸rna引物，然后将它们切换到Polε以开始 前导链复制和Polδ，用于开始复制数以百万计的冈崎片段中的每一个 落后的一股。B家族中剩下的成员是DNA聚合酶ζ(POLζ)，它是 跨损伤的DNA合成。目前对B家族DNA的理解仍然存在着显著的差距 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 复制分叉。这种全球协调的关键组件是模板：Primer的机制 在领先和滞后链的不对称合成期间从POLα切换到POLε和POLδ， 计算冈崎片段的长度，以及POLδ和POLε切换到生产性伸长。我们 发现POLδ的辅助B亚基也与POLζ的催化亚基形成络合物， 对病变搭桥过程中的聚合酶开关很重要。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性信息不足， 特别是对于整个聚合酶复合体，包括POLδ、POLε和POLζ，因为充分了解 分子结构对于设计有意义的功能分析是必不可少的。基于我们之前的 关于原始体及POLδ、POLε和POLζ成分的生产性研究，我们提出了一个新的方向 检查引物合成、从POLα到POLε的引物切换中紧密协调事件的调查 和POLδ，并切换到精确伸长模式。对于拟议的研究，我们将使用X光 结晶学和各种结构指导的生物化学和单分子实验。其中大多数都是 研究将使用体外重组的人类复制体进行。

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.