HELICASE ACTIVITY AND ITS ROLE IN TELOMERE AND TELOMERASE REGULATION

解旋酶活性及其在端粒和端粒酶调节中的作用

基本信息

批准号：
9892425
负责人：
Roberto Galletto
金额：
$ 5.93万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-08-01 至 2021-02-28
项目状态：
已结题

项目摘要

Abstract/Summary In this proposal we focus on the Pif1 sub-family of DNA helicases and test the hypothesis that one of their functions is to facilitate DNA replication across naturally occurring barriers. We showed that DNA-bound Rap1 is a barrier to the strand-displacement DNA synthesis activity of DNA polymerase  and that Pif1 displaces Rap1, allowing DNA replication across the barrier. We hypothesize that proteins bound to the DNA, at telomeres and non-telomeric sites, are displaced by Pif1 to facilitate DNA replication, and that this activity is fundamental and conserved among Pif1 homologues. G-quadruplexes and DNA hairpins can also be obstacles to DNA replication. Pif1 is thought to facilitate replication-fork progression by unwinding G-quadruplexes. However, little is known about this activity of Pif1 in the presence of single-stranded DNA binding proteins (SSBs). We hypothesize that the activity of Pif1 is needed to facilitate DNA replication across G- quadruplexes when SSBs cannot effectively melt the DNA secondary structure. Furthermore, we hypothesize that Pif1 stimulates DNA synthesis by DNA polymerase  across DNA hairpins. We showed that unwinding of dsDNA by a Pif1 monomer is balanced by a Pif1-dependent DNA rewinding activity. We hypothesize that under physiological conditions rewinding is inhibited by concomitant synthesis of DNA by polymerases. Mutations of Pif1 that affect the balance between unwinding and rewinding will allow us to determine the origin of this newly discovered property. To test these hypotheses, we propose three Aims: 1) determine whether protein obstacles that impede DNA replication are displaced by Pif1; 2) determine whether DNA secondary structures are unwound by Pif1 to facilitate DNA replication; 3) determine the mechanism underlying the Pif1-dependent DNA rewinding activity.

摘要/摘要在此提案中，我们专注于DNA解旋酶的PIF1亚家族，并检验了以下假设。他们的功能之一是促进自然发生的屏障的DNA复制。我们结果表明，结合DNA的RAP1是链置位置DNA合成活性的障碍 DNA聚合酶，PIF1置换RAP1，允许在屏障上复制DNA。我们假设蛋白质与DNA结合，端粒和非质体位点的蛋白质移位通过PIF1促进DNA复制，此活动是基本的，并且构成 PIF1同源物。 G-四链体和DNA发夹也可能是DNA复制的障碍。人们认为PIF1可以通过放松G四链体来促进复制前进。然而，在存在单链DNA结合蛋白的情况下，PIF1的这种活性知之甚少（SSB）。我们假设需要PIF1的活性来促进跨G-的DNA复制当SSB无法有效地融化DNA二级结构时，四链体。此外，我们假设PIF1刺激DNA聚合酶在DNA发夹中刺激DNA合成。我们表明，PIF1单体对DSDNA的放松是通过PIF1依赖性DNA平衡的倒带活动。我们假设在生理条件下，会抑制通过聚合酶伴有DNA的合成。影响PIF1的突变放松和倒带将使我们能够确定这种新发现的财产的起源。为了检验这些假设，我们提出了三个目的：1）确定蛋白质障碍是否存在阻碍DNA复制的pif1位移。 2）确定DNA是否次级通过PIF1解开结构，以促进DNA复制； 3）确定机制依赖PIF1的DNA倒带活性的基础。