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

• 批准号: 8897389
• 负责人: Roberto Galletto
• 金额: $ 28.73万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8897389
• 关键词: ATP phosphohydrolase; Affect; Binding; Binding Proteins; Biochemical; Biological Models; Cell Aging; Chromosomal Instability; Chromosomes; Complex; Coupled; DNA; Data; Dimerization; Enzymes; Excision; Goals; Knowledge; Link; Maintenance; Mission; Motor; Motor Activity; N-terminal; Nucleic Acids; Nucleoproteins; Nucleotides; Okazaki fragments; Play; Process; Property; Protein Binding; Proteins; Public Health; RNA-Directed DNA Polymerase; Regulation; Replication-Associated Process; Research; Ribosomal DNA; Role; Saccharomyces cerevisiae; Site; Study models; Telomerase; Telomerase Inhibitor; Telomerase inhibition; Telomere Maintenance; Testing; Yeasts; cofactor; ds-DNA; helicase; human disease; insight; telomere; telomere loss; translocase; tumorigenesis

DESCRIPTION (provided by applicant): The inter-conversion of telomeres between different functional states, defined by the composition of the bound proteins, is the fundamental step that dictates whether the chromosome end is recognized as a bona-fide end or incorrectly as a double-strand break. In recent years it has become evident that motor proteins (e.g. helicases) play a role in telomere regulation. Even in yeast, one of the best-studied model systems, how helicases function in telomere regulation is not fully understood. For example, it is not clear whether the major function of these motor proteins originate from their helicase activity and thus their ability to unwind double-stranded nucleic acid or from their ability to translocate along DNA and remove bound proteins. In S.cerevisiae, Pif1 helicase affects telomere function via displacement of the telomerase, the specialized reverse transcriptase responsible for extension of the G-rich strand of the telomere. A second helicase, Rrm3, has been implicated in telomere function. Rrm3 facilitates replication fork progression at >1000 chromosomal sites, including telomeres. A possible function of Rrm3 in replication fork progression at telomeres is the displacement of Rap1 bound to the duplex region of the telomere. Knowledge of the intrinsic biochemical properties of Pif1 and Rrm3 and how they are employed/altered to carry out their functions is necessary to elucidate how these motor proteins participate in telomere regulation. In Specific Aim 1 we will: A) Study the allosteric modulation by nucleotide cofactors of Pif1-DNA interactions. B) Determine which oligomeric state of Pif1 is responsible for its activity as a helicase or a translocase and how Pif1 catalyzes these activities. C) Test whether Pif1 inhibition of the telomerase originates from its helicase activity or its ability to displace subunits of the telomerase complex. In Specific Aim 2 we will: A) Test our hypothesis that the ATPase and helicase activities of Rrm3 are regulated by a sequence/domain within its N-terminal region. B) Test the hypothesis that PCNA binding to Rrm3 relieves the regulatory function of its N-terminal region. C) Test the hypothesis that interaction with PCNA leads to an active Rrm3 that is able to displace Rap1 from the telomere. These studies will allow us to determine how Pif1 and Rrm3 function and will provide unique insight on how these enzymes participate in telomere/telomerase regulation. We expect to define whether oligomerization of Pif1 leads to separation of its functions and thus to its specific role at telomeres. Also, we expect to determine how the activities of Rrm3 can be regulated to allow removal of proteins bound to double-stranded region of the telomere, thus facilitating replication of these sites.

描述（由申请人提供）：不同功能状态之间的端粒的会议（由结合蛋白的组成定义）是决定染色体端是识别为善良的端端还是错误地视为双重股骨突破的基本步骤。近年来，运动蛋白（例如解旋酶）在端粒调节中发挥作用，已经很明显。即使在酵母中，最有研究的模型系统之一，在端粒调节中的旋转酶的功能也不完全理解。例如，尚不清楚这些运动蛋白的主要功能是由它们的解旋酶活性起源，因此是否可以放松双链核酸或沿DNA转移并去除结合蛋白的能力。 在Cerevisiae中，PIF1解旋酶通过位移端粒酶（负责延伸端粒G富含G型链的专门逆转录酶）来影响端粒功能。第二个解旋酶RRM3与端粒功能有关。 RRM3促进在> 1000个染色体位点（包括端粒）的复制叉进展。 RRM3在端粒处的复制叉进程中的可能功能是RAP1与端粒的双工区域结合的位移。 必须了解PIF1和RRM3的内在生化特性以及如何使用/更改以执行其功能，这对于阐明这些运动蛋白如何参与端粒调节是必要的。在特定目的1中，我们将：a）研究PIF1-DNA相互作用的核苷酸辅因子的变构调节。 b）确定PIF1的低聚物状态对其作为解旋酶或易位酶的活性负责，以及PIF1如何催化这些活性。 c）测试PIF1抑制端粒酶是源自其解旋酶活性还是取代端粒酶复合物亚基的能力。在特定目的2中，我们将：a）检验我们的假设，即RRM3的ATPase和Helicase活性受其N末端区域内的序列/域调节。 b）检验以下假设：PCNA与RRM3结合可缓解其N末端区域的调节功能。 c）检验以下假设：与PCNA相互作用会导致活跃的RRM3，该RRM3能够从端粒中取代RAP1。 这些研究将使我们能够确定PIF1和RRM3的功能，并就这些酶如何参与端粒/端粒酶调节提供独特的见解。我们期望定义PIF1的低聚是否会导致其功能分离，从而使其在端粒中的特定作用。同样，我们希望如何调节RRM3的活性，以允许去除与端粒双链区域结合的蛋白质，从而促进这些位点的复制。