HELICASE ACTIVITY AND ITS ROLE IN TELOMERE AND TELOMERASE REGULATION

摘要/摘要 在这个提案中，我们重点关注DNA解旋酶的Pif1亚家族，并检验以下假设 它们的功能之一是促进跨越自然产生的障碍的DNA复制。我们 结果表明，DNA结合的Rap1是链置换DNA合成活性的障碍 DNA聚合酶和Pif1取代RAP1，允许dna复制跨越屏障。我们 假设与DNA结合的蛋白质在端粒和非端粒位置发生移位 通过Pif1促进DNA复制，并且这一活性是基本的，并且在 Pif1同源基因。G-四链体和DNA发夹也可能是DNA复制的障碍。 Pif1被认为通过解开G-四链体来促进复制-叉状进展。然而， 关于Pif1在单链DNA结合蛋白存在下的这种活性知之甚少 (SSB)。我们假设Pif1的活性是促进G-DNA复制所必需的。 当SSB不能有效地融化DNA二级结构时，就会产生四链。此外，我们 假设Pif1通过dna聚合酶跨dna发夹刺激dna合成。 我们发现，Pif1单体对dsDNA的解离与依赖于Pif1的DNA平衡 倒带活动。我们假设，在生理条件下，倒带受到抑制 伴随着聚合酶合成DNA。影响Pif1平衡的Pif1基因突变 解开和重绕将使我们能够确定这一新发现的财产的来源。 为了验证这些假说，我们提出了三个目标：1)确定蛋白质障碍 阻碍DNA复制的基因被Pif1取代；2)确定DNA是否次要 结构被Pif1解开以促进DNA复制；3)确定其机制 Pif1依赖的DNA倒绕活动的基础。

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.