DNA REPLICATION AND CHROMOSOME STRUCTURE IN YEAST

这个项目的长期目标是阐明 确保真核细胞染色体的忠实维护，使用酵母 以酿酒酵母为模型系统。这笔资金的总体目标是 这一时期是为了了解端粒复制是如何在酵母菌中调节的。 第一个主要目标是研究两个解旋酶，Pif1p和Rrm3p。这些 解旋酶彼此高度相似，是解旋酶的成员 从酵母到人类都很保守的亚科。Pif1p和Rrm3p都是 影响端粒，但方式不同。Rrm3p似乎在晚些时候采取行动 端粒复制中的一步，被认为对产生 端粒酶的底物，从而促进端粒酶。Pif1p似乎在发挥作用 Rrm3p的下游，其作用抑制端粒酶。目标1描述了一个 一系列的遗传、生化和DNA结构研究，以了解 Pif1p和Rrm3p调节端粒酶。野生型和突变型重组Pif1p和 RRM3P将被提纯并用于确定底物的偏好。两者都有 将在体外测试蛋白质对端粒酶活性的影响。在……里面 对突变等位基因的活体分析将确定解旋酶是否具有 Pif1p和Rrm3p负责它们对端粒复制的影响。 染色质免疫沉淀(CHIP)将确定Pif1p和/或Rrm3p是否 在物理上与端粒DNA相关。基因方法将识别 与Rrm3p功能重叠的基因，并确定是否缺乏 Rrm3p触发端粒特异性检查点。体外和体内方法 将确定Pif1p是否通过其核溶解降解来抑制端粒酶 底物。第二个目标是了解一种非常不同类型的 端粒酶调节因子，一种端粒结构蛋白，控制着 端粒转化为端粒酶。Rif1p和Rif2p是端粒结合蛋白，作用于 协同作用以限制端粒的延长。芯片将被用来确定 如果Rif蛋白通过细胞周期或细胞周期调节端粒DNA对端粒酶的访问 端粒长度依赖的结合。Rif蛋白对复制的影响 端粒的计时与端粒酶非依赖性重组端粒 还将确定维护情况。第三个目标是确定其他 突变或过度表达促进端粒酶介导的愈合的基因 断裂的染色体。越来越多的证据表明，端粒复制已经 对衰老和癌症都有影响。作为酵母端粒DNA和支配 其性质从酵母到人类在功能和/或结构上是保守的， 了解酵母中的端粒调节可能与遗传不稳定性有关 在人类身上。

The long term goal of this project is to elucidate the process that ensures faithful maintenance of eukaryotic chromosomes, using the yeast Saccharomyces cerevisiae as a model system. The general goal of this funding period is to understand how telomere replication is regulated in Saccharomyces. The first and major aim focuses on two helicases, Pif1p and Rrm3p. These helicases are highly similar to each other and are members of a helicase sub-family that is conserved from yeast to humans. Pif1p and Rrm3p both influence telomeres but not in the same way. Rrm3p appears to act in a late step in telomere replication that is proposed to be important for generating a substrate for telomerase and hence promotes telomerase. Pif1p appears to act down stream of Rrm3p, and its actions inhibit telomerase. Aim 1 describes a series of genetic, biochemical, and DNA structural studies to understand how Pif1p and Rrm3p regulate telomerase. Wild type and mutant recombinant Pif1p and Rrm3p will be purified and used to determine substrate preferences. Both proteins will be tested for the effects on telomerase activity in vitro. In vivo analysis of the mutant alleles will determine if the helicase functions of Pif1p and Rrm3p are responsible for their effects on telomere replication. Chromatin immuno-precipitation (ChIP) will determine if Pif1p and/or Rrm3p are physically associated with telomeric DNA. Genetic approaches will identify genes that have overlapping functions with Rrm3p and to determine if lack of Rrm3p triggers a telomere-specific checkpoint. In vitro and in vivo approaches will determine if Pif1p inhibits telomerase by nucleolytic degradation of its substrate. The second aim is to understand how a very different type of telomerase regulator, a telomere structural protein, governs access of telomeres to telomerase. Rif1p and Rif2p are telomere binding proteins that act synergistically to limit telomere lengthening. ChIP will be used to determine if Rif proteins regulate access of telomeric DNA to telomerase by cell cycle or telomere length dependent binding. The effects of Rif proteins on replication timing of telomeres and on telomerase-independent, recombinational telomere maintenance will also be determined. The third aim is to identify additional genes whose mutation or over-expression increases telomerase mediated healing of broken chromosomes. There is increasing evidence that telomere replication has effects on both aging and cancer. As yeast telomeric DNA and the proteins that govern its properties are functionally and/or structurally conserved from yeast to humans, understanding telomere regulation in yeast is likely to be relevant to genetic instability in humans.