Human DNA Replication Machines: Structure-function Studies

人类 DNA 复制机器：结构功能研究

• 批准号: 10396028
• 负责人: Tahir H Tahirov
• 金额: $ 45.75万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10396028
• 关键词: Biochemical; Biological Assay; Bypass; Catalytic Domain; Complex; Crystallization; DNA Primase; DNA Primers; DNA biosynthesis; DNA polymerase zeta; DNA replication fork; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Deoxyribonucleotides; Drug Design; Drug Targeting; Event; Family; Future; Genome; Health; Human; In Vitro; Investigation; Knowledge; Length; Lesion; Life; Malignant Neoplasms; Medicine; Molecular Structure; Okazaki fragments; Patients; Polymerase; Process; Proteins; RNA primers; Structure; Transact; Viral; X-Ray Crystallography; Yeasts; base; design; disease diagnosis; experimental study; human DNA; member; mutant; reconstitution; single molecule; virtual

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.

项目概要/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于疾病的未来进展至关重要 诊断、药物设计和患者治疗。人类 DNA 复制的大部分是由 B 家族完成的 DNA 聚合酶 Polδ 和 Polε。然而，如果没有 DNA 引物，Polδ 和 Polε 就无法开始合成。到 为了规避这个问题，一种称为引物酶的特殊 RNA 聚合酶会生成初始引物。然后，一个 专用 B 家族 DNA 聚合酶 α (Polα) 与引物酶（称为 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 引导链复制和 Polδ 用于开始复制数百万个冈崎片段中的每一个 滞后链。 B 家族的其余成员是 DNA 聚合酶 ze (Pol )，它是 跨损伤DNA合成。目前对 B 家族 DNA 的理解仍存在重大差距 聚合酶的功能，特别是关于紧密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 template:primer 的机制 在前导链和滞后链的不对称合成过程中从 Polα 切换到 Polε 和 Polδ， 计算冈崎片段的长度，以及 Polδ 和 Polε 到有效伸长的转换。我们 发现 Polδ 的辅助 B 亚基也与 Polδ 的催化亚基形成复合物，该复合物 对于病变旁路期间的聚合酶开关很重要。然而，这种开关的机制仍然存在 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 Polδ、Polε 和 Polζ，因为充分了解 分子结构对于设计有意义的功能测定至关重要。根据我们之前的 通过对引发体和 Polδ、Polε 和 Polζ 组成部分的富有成效的研究，我们提出了一个新的方向 检查引物合成、引物从 Polα 到 Polε 的切换中紧密协调的事件的调查 和 Polδ，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 晶体学和各种结构引导的生化和单分子实验。其中大部分 研究将使用体外重组的人类复制体进行。

项目成果

Translesion activity of PrimPol on DNA with cisplatin and DNA-protein cross-links.

• DOI: 10.1038/s41598-021-96692-y
• 发表时间: 2021-09-02
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Boldinova EO;Yudkina AV;Shilkin ES;Gagarinskaya DI;Baranovskiy AG;Tahirov TH;Zharkov DO;Makarova AV
• 通讯作者: Makarova AV

Insight into RNA-DNA primer length counting by human primosome.

• DOI: 10.1093/nar/gkac492
• 发表时间: 2022-06-24
• 期刊: NUCLEIC ACIDS RESEARCH
• 影响因子: 14.9
• 作者: Baranovskiy, Andrey G.;Lisova, Alisa E.;Morstadt, Lucia M.;Babayeva, Nigar D.;Tahirov, Tahir H.
• 通讯作者: Tahirov, Tahir H.

DNA primase large subunit is an essential plant gene for geminiviruses, putatively priming viral ss-DNA replication.

• DOI: 10.3389/fpls.2023.1130723
• 发表时间: 2023
• 期刊: Frontiers in plant science
• 影响因子: 5.6

Human DNA polymerase α has a strong mutagenic potential at the initial steps of DNA synthesis.

• DOI: 10.1093/nar/gkac1101
• 发表时间: 2022-11-28
• 期刊: NUCLEIC ACIDS RESEARCH
• 影响因子: 14.9
• 作者: Lisova, Alisa E.;Baranovskiy, Andrey G.;Morstadt, Lucia M.;Babayeva, Nigar D.;Tahirov, Tahir H.
• 通讯作者: Tahirov, Tahir H.

The role of catalytic and regulatory domains of human PrimPol in DNA binding and synthesis.

• DOI: 10.1093/nar/gkad507
• 发表时间: 2023-08-11
• 期刊: Nucleic acids research
• 影响因子: 14.9