DNA Replication and Chromosome Structure in Yeast

酵母中的 DNA 复制和染色体结构

• 批准号: 8389594
• 负责人: VIRGINIA A. ZAKIAN
• 金额: $ 58.87万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-07-01 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8389594
• 关键词: Abbreviations; Affect; Base Pairing; Binding; Binding Sites; Biological Assay; Cells; ChIP-on-chip; Chromatin; Chromosomal Rearrangement; Chromosome Breakage; Chromosome Structures; Chromosomes; DNA; DNA Damage; DNA Structure; DNA biosynthesis; Ensure; Event; Family; Fission Yeast; Frequencies; Funding; G-Quartets; Gene Mutation; Genes; Genetic; Genome; Genome Stability; Genomic Instability; Goals; Grant; Human; Hydroxyl Radical; In Vitro; Inherited; Lead; Length; Maintenance; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Modeling; Mutation; Names; Nucleic Acids; Open Reading Frames; Plasmids; Polymerase; Polymerase Chain Reaction; Precipitation; Premature aging syndrome; Process; Property; Protein Family; Proteins; Recombinant DNA; Ribosomal DNA; Role; Saccharomyces cerevisiae; Site; Structure; Telomerase; Testing; Urea; Work; Yeasts; cell type; chromosome replication; directed evolution; genome-wide; helicase; human disease; in vivo; insight; member; mutant; preference; protein complex; public health relevance; recombinational repair; research study; telomere; two-dimensional

DESCRIPTION (provided by applicant): The long term goal of this grant is to contribute to the understanding of mechanisms responsible for the faithful replication of eukaryotic chromosomes, using yeasts as models. The four aims concern three members of the Pif1 family of DNA helicases, a virtually ubiquitous eukaryotic helicase family that is named for its prototypical member the S. cerevisiae Pif1. The first aim will determine how the S. cerevisiae Pif1 inhibits telomerase- mediated telomere extension by asking if Pif1 inhibits the frequency and/or extent of telomere lengthening and to determine if its activity is regulated by telomere length. The second aim extends our recent discovery that the S. cerevisiae Pif1 is a potent unwinder of G-quadruplex DNA (G4), very stable four-stranded DNA structures that are held together by G-G base pairs. We will use genome wide approaches to identify Pif1 binding sites and sites of DNA damage in Pif1-depeleted cells to see if Pif1 acts preferentially at G4 sites. A variety of genetic and physical assays will be used to determine if sequences that can form G4 structures in vitro are selectively destabilized in pif1 mutant cells and if so, if this instability is correlated with fork stalling and/or breakage at G4 sites. These experiments will help resolve controversies as to whether G4 structures form in vivo. The third aim will continue our studies to determine the essential role(s) of the S. pombe Pfh1 helicase in chromosome replication. To identify Pfh1 targets, we will again use genome wide approaches, which will identify Pfh1 binding sites, as well as sites of chromosome breakage upon Pfh1 depletion. Physical and genetic assays will be used to determine if Pfh1 depletion results in fork stalling and/or breakage at hard to replicate sites and/or at sites identified in the genome wide analyses. Pfh1 interacting proteins will be identified by mass spectrometry with the goal that their identities will help determine the specific DNA transactions in which Pfh1 is engaged. The fourth aim describes work to continue our analysis of the S. cerevisiae Rrm3 helicase, which has the unique property of promoting fork progression through stable protein complexes. To circumvent difficulties purifying full length Rrm3 for in vitro studies, we will use a genetic strategy to identify mutations in the amino terminus of Rrm3 that render it easy to purify without affecting its in vivo functions. We will use purified Rrm3 to determine its preferred nucleic acid substrates and to test its ability to displace protein complexes from DNA in vitro. A genetic approach is described to identify proteins that either assist Rrm3 or substitute for it during replication through stable protein complexes. Because helicases are essential for DNA replication, repair and recombination, it is not surprising that their mutation can lead to human diseases characterized by genome instability, such as premature aging, and cancer. In yeasts, Pif1 family proteins have important and so far unique properties in DNA replication. These studies will lay the groundwork for the identification and analysis of human proteins with similar functions.

描述（由申请人提供）：这项资助的长期目标是促进对真核染色体忠实复制机制的理解，使用酵母作为模型。这四个目标涉及 DNA 解旋酶 Pif1 家族的三个成员，这是一个几乎无处不在的真核解旋酶家族，以其原型成员酿酒酵母 Pif1 命名。第一个目标是通过询问 Pif1 是否抑制端粒延长的频率和/或程度来确定酿酒酵母 Pif1 如何抑制端粒酶介导的端粒延长，并确定其活性是否受端粒长度调节。第二个目标扩展了我们最近的发现，即酿酒酵母 Pif1 是 G-四链体 DNA (G4) 的有效解旋器，G4 是由 G-G 碱基对结合在一起的非常稳定的四链 DNA 结构。我们将使用全基因组方法来识别 Pif1 缺失细胞中的 Pif1 结合位点和 DNA 损伤位点，以了解 Pif1 是否优先在 G4 位点发挥作用。将使用各种遗传和物理测定来确定可在体外形成G4结构的序列是否在pif1突变细胞中选择性地不稳定，如果是的话，这种不稳定是否与G4位点的叉停顿和/或断裂相关。这些实验将有助于解决关于 G4 结构是否在体内形成的争议。第三个目标是继续我们的研究，以确定粟酒裂殖酵母 Pfh1 解旋酶在染色体复制中的重要作用。为了确定 Pfh1 靶标，我们将再次使用全基因组方法，该方法将识别 Pfh1 结合位点以及 Pfh1 耗尽时的染色体断裂位点。物理和遗传测定将用于确定 Pfh1 耗尽是否导致叉在难以复制位点和/或在全基因组分析中确定的位点处停滞和/或断裂。 Pfh1 相互作用蛋白将通过质谱法进行鉴定，其目的是帮助确定 Pfh1 参与的特定 DNA 事务。第四个目标描述了继续分析酿酒酵母 Rrm3 解旋酶的工作，该解旋酶具有通过稳定的蛋白质复合物促进分叉进展的独特特性。为了避免在体外研究中纯化全长 Rrm3 的困难，我们将使用遗传策略来识别 Rrm3 氨基末端的突变，使其易于纯化而不影响其体内功能。我们将使用纯化的 Rrm3 来确定其首选的核酸底物，并测试其在体外置换 DNA 中蛋白质复合物的能力。描述了一种遗传方法来识别在复制过程中通过稳定的蛋白质复合物辅助 Rrm3 或替代 Rrm3 的蛋白质。由于解旋酶对于 DNA 复制、修复和重组至关重要，因此它们的突变可能导致以基因组不稳定为特征的人类疾病，例如过早衰老和癌症，也就不足为奇了。在酵母中，Pif1 家族蛋白在 DNA 复制中具有重要且迄今为止独特的特性。这些研究将为鉴定和分析具有相似功能的人类蛋白质奠定基础。