DNA REPLICATION AND CHROMOSOME STRUCTURE IN YEAST

这项资助的长期目标是阐明确保真核生物忠实维持的过程。 染色体，使用酵母酿酒酵母作为模型系统。端粒， 染色体，是必不可少的稳定性和完整性的酵母染色体。酵母染色体以约300 bp的 Ci-3A/TGi_3DNA。酵母端粒DNA通常由逆转录酶端粒酶合成，尽管 重组可以在缺乏端粒酶的细胞中维持酵母端粒DNA。本资助期的总目标是 了解酵母中端粒复制是如何调节的。第一个也是主要的目标集中在两个解旋酶， Pif Ip和RrmSp。这些解旋酶彼此高度相似，并且是解旋酶亚家族的成员， 从酵母菌到人类都保存了下来PifIp和RrmSp都影响端粒，但方式不同。Rrm 3 p似乎 在端粒复制的后期步骤中起作用，这被认为对产生端粒酶底物很重要， 从而促进端粒酶。Piflp似乎作用于Rrm 3 p的下游，并且其作用抑制端粒酶。要求1 描述了一系列遗传、生化和DNA结构研究，以了解Piflp和Rrm 3如何调节 端粒酶将纯化野生型和突变型重组Piflp和Rrm 3 p并用于测定底物 喜好这两种蛋白质将在体外测试对端粒酶活性的影响。突变体的体内分析 等位基因将决定Piflp和Rrm 3 p的解旋酶功能是否负责它们对端粒的影响 复制的染色质免疫沉淀（ChIP）将确定Piflp和/或Rrm 3 p是否物理相关 端粒DNA遗传学方法将鉴定与Rrm 3 p具有重叠功能的基因， 确定缺少Rrm 3 p是否触发端粒特异性检查点。体外和体内方法将确定 Piflp通过其底物的溶核降解来抑制端粒酶。第二个目标是了解一个非常 不同类型的端粒酶调节剂，端粒结构蛋白，控制端粒对端粒酶的接近。Rif Ip 和Rif 2 p是端粒结合蛋白，其协同作用以限制端粒延长。 确定Rif蛋白是否通过细胞周期或端粒长度依赖性调节端粒DNA对端粒酶的访问 约束力Rif蛋白对端粒复制时间的影响以及对端粒酶非依赖性重组 还将确定端粒维持。第三个目标是确定其他基因的突变或过度- 表达增加端粒酶介导的断裂染色体的愈合。越来越多的证据表明端粒 复制对衰老和癌症都有影响。作为酵母端粒DNA和蛋白质，管理其属性， 从酵母到人类在功能上和/或结构上保守，理解酵母中的端粒调节可能会 与人类基因的不稳定性有关。

The long term goal of this grant is to elucidate the processes that ensure the faithful maintenance of eukaryotic chromosomes, using the yeast Saccharomyces cerevisiae as a model system. Telomeres, the physicalends of the chromosome, are essential for the stability and integrity of yeast chromosomes. Yeast chromosomes end in -300 bps of Ci-3A/TGi_3 DNA. Yeast telomeric DNA is normally synthesized by the reverse transcriptase telomerase, although recombination can maintain yeast telomeric DNA in cells lacking telomerase. 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, Pif Ip and RrmSp. These helicases are highly similar to each other and are members of a helicase sub-family that is conserved from yeast to humans. PifIp and RrmSp 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. Piflp appears to act downstream of Rrm3p, and its actions inhibit telomerase. Aim 1 describes a series of genetic, biochemical, and DNA structural studies to understandhow Piflp and Rrm3pregulate telomerase. Wild type and mutant recombinant Piflp and Rrm3p will be purified and used to determinesubstrate preferences. Both proteins will be tested for the effects on telomerase activity in vitro. In vivo analysisof the mutant alleles will determine if the helicase functions of Piflp and Rrm3p are responsible for their effects on telomere replication. Chromatin immuno-precipitation (ChIP) will determine if Piflp 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 Piflp inhibits telomerase by nucleolytic degradation of its substrate. The second aim is to understandhow a very different type of telomerase regulator, a telomere structural protein, governs access of telomeres to telomerase. Rif Ip 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 lengthdependent 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.