DNA Replication and Chromosome Structure in Yeast

描述(由申请者提供)：这项资助的长期目标是以酵母菌为模型，促进对真核染色体忠实复制机制的理解。这四个目标涉及DNA解旋酶Pif1家族的三个成员，Pif1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。第一个目的将通过询问Pif1是否抑制端粒延长的频率和/或程度来确定Pif1是如何抑制端粒酶介导的端粒延长的，并确定其活性是否受端粒长度的调节。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DNA(G4)的有效解旋器，G-四链DNA是非常稳定的四链DNA结构，通过G-G碱基对连接在一起。我们将使用全基因组方法来确定Pif1结合部位和Pif1去皮细胞中的DNA损伤部位，以确定Pif1是否优先在G4部位起作用。各种遗传和物理分析将被用来确定在体外可以形成G4结构的序列在pif1突变细胞中是否选择性地不稳定，如果是，这种不稳定是否与G4位点的叉子失速和/或断裂有关。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。第三个目标将继续我们的研究，以确定S.pombe Pfh1解旋酶在染色体复制中的重要作用(S)。为了识别Pfh1靶点，我们将再次使用全基因组方法，这将识别Pfh1结合部位，以及Pfh1缺失时染色体断裂的部位。物理和遗传分析将被用来确定Pfh1耗尽是否导致难以复制的位置和/或在全基因组分析中确定的位置的分叉停滞和/或断裂。Pfh1相互作用的蛋白质将通过质谱学进行鉴定，目标是它们的身份将有助于确定Pfh1参与的特定DNA交易。第四个目标描述了继续我们对酿酒酵母Rrm3解旋酶的分析工作，该解旋酶具有通过稳定的蛋白质复合体促进分叉进展的独特性质。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确定RRM3的氨基末端的突变，这些突变使其易于纯化，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，并测试其在体外从DNA中置换蛋白质复合体的能力。描述了一种通过稳定的蛋白质复合体在复制过程中识别辅助RRM3或替代RRM3的蛋白质的遗传方法。由于解旋酶对DNA复制、修复和重组是必不可少的，因此它们的突变会导致以基因组不稳定为特征的人类疾病也就不足为奇了，例如过早衰老和癌症。在酵母中，Pif1家族蛋白在DNA复制中具有重要而独特的特性。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。 与公共卫生相关：DNA解旋酶在DNA复制、修复和重组中起着关键作用。因此，它们的突变会导致基因组不稳定，并导致癌症和早衰等遗传性人类疾病也就不足为奇了。这笔赠款描述了确定真核细胞Pif1家族解旋酶对基因组稳定性做出贡献的机制的实验，这些解旋酶在染色体复制中具有独特的特性。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。

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. PUBLIC HEALTH RELEVANCE: DNA helicases have critical roles in DNA replication, repair and recombination. Therefore, it is not surprising that their mutation can lead to genome instability and inherited human diseases such as cancer and premature aging. This grant describes experiments to determine the mechanisms by which the eukaryotic Pif1 family helicases, which have unique properties in chromosome replication, contribute to genome stability. These studies will lay the groundwork for the identification and analysis of human proteins with similar functions.