Enzyme Interactions at the DNA Replication Fork

DNA 复制叉上的酶相互作用

• 批准号: 7149439
• 负责人: Judith L CAMPBELL
• 金额: $ 29.27万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-20 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7149439
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA replication; DNA replication origin; affinity chromatography; cytogenetics; endodeoxyribonuclease; enzyme mechanism; enzyme reconstitution; enzyme substrate; functional /structural genomics; fungal proteins; guanine nucleotides; helicase; protein isoforms; protein protein interaction; telomerase; telomere

DESCRIPTION (provided by applicant): Fidelity of copying of the genome during DNA replication is maintained at a robust level by a poorly understood network of intersecting pathways. Specific mechanisms by which these pathways protect the genome remain uncharacterized due to the complexity of the underlying processes at the replication fork and their regulation. A major challenge is to understand if and how the replication apparatus coordinates the genome maintenance machineries. Recently, we have used global genetic interaction screens (SGA) and have defined an elaborate network of replication, repair, and regulatory (checkpoint and cell cycle) genes that we propose preserves the integrity of the lagging strand at the replication fork. DNA polymerase delta, FEN1 nuclease, and the essential replication helicase/nuclease Dna2 are key hubs in this network of Okazaki fragment synthesis and processing (OFP) enzymes, as are the Sgs1, Rrm3, Pif1, and Srs2 helicases. The pathways in the network define major avenues for guarding the genome and have implications for understanding of diseases such as cancer and aging that may derive from genome instability. Two major specific insights have been gained from analysis of the network and dictate our new directions: (1) We have found that the requirement for Dna2 protein for viability in yeast can be bypassed by deletion of another helicase, Pif1. Genetic evidence further suggests strong interaction of both Dna2 and Pif1 with DNA polymerase delta. We will probe the contribution of Pif1 to accurate lagging strand replication, using biochemical reconstitution, emphasizing the contribution of Pif1 to the well-characterized reactions of Dna2, FEN1, and pol delta on model substrates mimicking OFP intermediates. (2) We have found that deletion of DNA2 suppresses the excessive telomere elongation observed in pif1 mutants, adding to significant previous evidence that Dna2 functions at telomeres. We will study lagging strand synthesis on telomeric DNAs as well as interaction of Dna2 with various G-quadruplex Structures that may occur at telomeres (and elsewhere in the genome). Additional possible roles for Dna2 at telomeres will be tested, such as a role in recruiting telomerase and in degrading uncapped telomeres. Telomere length in dna2 mutants will be examined and compared with length in other mutants affecting telomere homeostasis.

描述（由申请人提供）：DNA 复制过程中基因组复制的保真度通过人们知之甚少的交叉途径网络维持在稳健的水平。由于复制叉的基础过程及其调节的复杂性，这些途径保护基因组的具体机制仍然未知。一个主要的挑战是了解复制装置是否以及如何协调基因组维护机制。最近，我们使用了全局遗传相互作用筛选（SGA），并定义了一个复杂的复制、修复和调节（检查点和细胞周期）基因网络，我们建议保留复制叉处滞后链的完整性。 DNA 聚合酶 delta、FEN1 核酸酶和必需的复制解旋酶/核酸酶 Dna2 是冈崎片段合成和加工 (OFP) 酶网络中的关键枢纽，Sgs1、Rrm3、Pif1 和 Srs2 解旋酶也是如此。网络中的通路定义了保护基因组的主要途径，并对理解可能源于基因组不稳定的疾病（例如癌症和衰老）具有重要意义。从网络分析中获得了两个主要的具体见解，并决定了我们的新方向：（1）我们发现，通过删除另一种解旋酶 Pif1，可以绕过酵母中 Dna2 蛋白的活力要求。遗传证据进一步表明 Dna2 和 Pif1 与 DNA 聚合酶 delta 存在强烈的相互作用。我们将使用生化重构来探讨 Pif1 对精确滞后链复制的贡献，强调 Pif1 对模仿 OFP 中间体的模型底物上的 Dna2、FEN1 和 pol delta 的良好表征反应的贡献。 (2) 我们发现 DNA2 的缺失抑制了 pif1 突变体中观察到的端粒过度伸长，这为 Dna2 在端粒发挥作用的先前证据提供了重要证据。我们将研究端粒 DNA 上的滞后链合成以及 Dna2 与端粒（以及基因组其他地方）可能发生的各种 G 四链体结构的相互作用。将测试 Dna2 在端粒上的其他可能作用，例如招募端粒酶和降解未加帽端粒的作用。将检查 dna2 突变体中的端粒长度，并将其与影响端粒稳态的其他突变体中的长度进行比较。