Enzyme Interactions at the DNA Replication Fork

描述(由申请人提供)：在DNA复制过程中，基因组复制的保真度通过一个鲜为人知的交叉路径网络保持在一个稳健的水平。由于复制分叉的基本过程及其调控的复杂性，这些途径保护基因组的具体机制仍未确定。一个主要的挑战是了解复制设备是否以及如何协调基因组维护机制。最近，我们使用了全球遗传相互作用屏幕(SGA)，并定义了一个复杂的复制、修复和调控(检查点和细胞周期)基因网络，我们认为这些基因可以保持复制叉处滞后链的完整性。DNA聚合酶Delta、FEN1核酸酶和必需复制解旋酶/核酸酶DNA2是Okazaki片段合成和处理(OFP)酶网络中的关键枢纽，SGS1、RrM3、Pif1和Srs2解旋酶也是如此。该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，如癌症和衰老具有意义。通过对该网络的分析，我们获得了两个主要的具体见解，并指示了我们的新方向：(1)我们发现，通过删除另一种解旋酶Pif1，可以绕过酵母中生存所需的DNA2蛋白。遗传证据进一步表明，DNA2和Pif1都与DNA聚合酶Delta有很强的相互作用。我们将利用生化重组来探索Pif1对准确滞后链复制的贡献，强调Pif1对DNA2、FEN1和PolDelta在模拟OFP中间体的模型底物上的良好特征反应的贡献。(2)我们发现DNA2的缺失抑制了在pif1突变体中观察到的过度端粒延长，进一步证明了DNA2在端粒中起作用。我们将研究端粒DNA的滞后链合成，以及DNA2与端粒(和基因组其他地方)可能出现的各种G-四链结构的相互作用。DNA2在端粒上的其他可能作用将被测试，例如在招募端粒酶和降解无上限端粒方面的作用。DNA2突变体的端粒长度将被检测，并与其他影响端粒稳态的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.