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）：通过较知的相交途径网络将DNA复制过程中基因组复制的保真度保持在强大的水平上。这些途径保护基因组的特定机制由于复制叉及其调节的基础过程的复杂性而保持未表征。一个主要的挑战是了解复制机构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），并定义了我们提出的精心复制，修复和调节（检查点和细胞周期）基因的精细网络，可以保留在复制叉处滞后链的完整性。 DNA聚合酶三角洲，FEN1核酸酶和必需复制酶/核酸酶DNA2与SGS1，RRM3，PIF1和SRS2 Helicases中的Okazaki片段合成和（OFP）酶一样，是Okazaki片段合成和（OFP）酶的关键枢纽。网络中的途径定义了守护基因组的主要途径，并对可能源自基因组不稳定性的癌症和衰老等疾病的理解具有影响。从对网络的分析中获得了两个主要的特定见解，并决定了我们的新方向：（1）我们发现，DNA2蛋白在酵母中生存能的需求可以通过缺失另一个解旋酶PIF1来绕过。遗传证据进一步表明DNA2和PIF1与DNA聚合酶三角洲的相互作用很强。我们将使用生化重构探测PIF1对准确的滞后链复制的贡献，强调PIF1对模型底物的DNA2，FEN1和POL Delta对模型底物的良好特征反应的贡献。 （2）我们发现DNA2的缺失抑制了PIF1突变体中观察到的过度端粒伸长，这增加了先前的重要证据，即DNA2在端粒中的功能。我们将研究端粒DNA上的滞后合成以及DNA2与可能发生在端粒（以及基因组中其他地方）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，例如在募集端粒酶和降解无封闭端粒中的作用。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的长度进行比较。