Enzyme Interactions at the DNA Replication Fork

描述（由申请人提供）：DNA复制期间基因组复制的保真度通过对交叉途径网络知之甚少而保持在稳健水平。这些途径保护基因组的具体机制仍然没有表征，这是由于复制叉及其调控的基础过程的复杂性。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，我们已经使用了全球遗传相互作用屏幕（SGA），并已定义了一个精心制作的复制，修复和监管（检查点和细胞周期）的基因，我们建议保留在复制叉滞后链的完整性网络。DNA聚合酶delta、FEN 1核酸酶和必需的复制解旋酶/核酸酶Dna 2是冈崎片段合成和加工（OFP）酶网络中的关键枢纽，Sgs 1、Rrm 3、Pif 1和Srs 2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性的疾病（如癌症和衰老）具有意义。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Pif 1，可以绕过Dna 2蛋白在酵母中的生存能力。遗传学证据进一步表明DNA 2和Pif 1与DNA聚合酶δ的强相互作用。我们将探测Pif 1的贡献，准确滞后链复制，使用生化重建，强调Pif 1的贡献，以及表征的反应Dna 2，FEN 1，和POL δ上的模型基板模仿OFP中间体。(2)我们已经发现，DNA 2的缺失抑制pif 1突变体中观察到的过度端粒伸长，增加了以前的证据表明，DNA 2在端粒的功能。我们将研究端粒DNA上的滞后链合成，以及DNA 2与可能发生在端粒（和基因组其他地方）的各种G-四链体结构的相互作用。DNA 2在端粒的其他可能作用将被测试，例如在招募端粒酶和降解未加帽的端粒中的作用。dna 2突变体的端粒长度将被检测并与影响端粒稳态的其他突变体的长度进行比较。

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.