DNA Replication and Chromosome Structure in Yeast

描述（由申请人提供）：该基金的长期目标是以酵母为模型，促进对真核生物染色体忠实复制机制的理解。这四个目标涉及DNA解旋酶Pif 1家族的三个成员，这是一个几乎普遍存在的真核解旋酶家族，以其原型成员S.酿酒酵母Pif 1.第一个目标将决定S。通过询问Pif 1是否抑制端粒延长的频率和/或程度并确定其活性是否受端粒长度调节，来确定酿酒酵母Pif 1抑制端粒酶介导的端粒延长。第二个目标扩展了我们最近的发现，S。酿酒酵母Pif 1是G-四链体DNA（G4）的有效解旋器，G4是通过G-G碱基对保持在一起的非常稳定的四链DNA结构。我们将使用全基因组的方法来确定Pif 1结合位点和Pif 1-depeled细胞的DNA损伤位点，看看Pif 1是否优先作用于G4位点。将使用各种遗传和物理测定来确定在体外可以形成G4结构的序列是否在pif 1突变细胞中被选择性地去稳定化，如果是这样，这种不稳定性是否与G4位点处的叉停滞和/或断裂相关。这些实验将有助于解决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的基本作用。pombe Pfh 1解旋酶在染色体复制中的作用。为了鉴定Pfh 1靶标，我们将再次使用全基因组方法，其将鉴定Pfh 1结合位点以及Pfh 1耗尽后染色体断裂的位点。将使用物理和遗传测定来确定Pfh 1消耗是否导致难以复制位点和/或全基因组分析中鉴定的位点处的叉停滞和/或断裂。Pfh 1相互作用的蛋白质将通过质谱鉴定，其目标是它们的身份将有助于确定Pfh 1参与的特定DNA交易。第四个目标描述了我们继续分析S。酿酒酵母Rrm 3解旋酶，其具有通过稳定的蛋白质复合物促进叉进展的独特性质。为了避免在体外研究中纯化全长Rrm 3的困难，我们将使用遗传策略来鉴定Rrm 3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Rrm 3来确定其优选的核酸底物，并测试其在体外从DNA中置换蛋白质复合物的能力。描述了一种遗传方法，以确定蛋白质，要么协助Rrm 3或取代它在复制过程中通过稳定的蛋白质复合物。由于解旋酶对于DNA复制、修复和重组是必不可少的，因此它们的突变可以导致以基因组不稳定为特征的人类疾病，如过早衰老和癌症，这并不奇怪。在酵母中，Pif 1家族蛋白在DNA复制中具有重要且独特的性质。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.