HELICASE ACTIVITY AND ITS ROLE IN TELOMERE AND TELOMERASE REGULATION

描述（由申请人提供）：端粒在不同功能状态之间的相互转换，由结合蛋白的组成定义，是决定染色体末端是否被识别为真实末端或错误地识别为双链断裂的基本步骤。近年来，运动蛋白（如解旋酶）在端粒调控中发挥作用已经变得很明显。即使在酵母中，解旋酶是如何在端粒调控中起作用的，这是研究得最好的模型系统之一。例如，目前尚不清楚这些运动蛋白的主要功能是源于它们的解旋酶活性，从而能够解开双链核酸，还是源于它们沿DNA易位并去除结合蛋白的能力。在酿酒酵母中，Pif1解旋酶通过端粒酶的位移影响端粒功能，端粒酶是一种专门的逆转录酶，负责端粒富含g的链的延伸。另一种解旋酶Rrm3与端粒功能有关。Rrm3促进了包括端粒在内的1000个染色体位点的复制叉进展。Rrm3在端粒复制叉进程中的一个可能功能是端粒双工区Rap1的移位。了解Pif1和Rrm3的内在生化特性以及它们如何被利用/改变以执行其功能对于阐明这些运动蛋白如何参与端粒调节是必要的。在具体目标1中，我们将：A)研究核苷酸辅因子对Pif1-DNA相互作用的变构调节。B)确定Pif1的哪种寡聚状态负责其作为解旋酶或转位酶的活性，以及Pif1如何催化这些活性。C)测试Pif1对端粒酶的抑制是源于其解旋酶活性还是其取代端粒酶复合物亚基的能力。在具体目标2中，我们将：A)验证我们的假设，即Rrm3的atp酶和解旋酶活性受其n端区域内的序列/结构域调节。B)验证PCNA结合Rrm3解除其n端调控功能的假设。C)验证与PCNA相互作用导致活性Rrm3能够从端粒中取代Rap1的假设。这些研究将使我们能够确定Pif1和Rrm3的功能，并将为这些酶如何参与端粒/端粒酶调节提供独特的见解。我们希望确定Pif1的寡聚化是否导致其功能分离，从而确定其在端粒中的特定作用。此外，我们希望确定如何调节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。

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. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because chromosome instability, oncogenesis and cellular senescence are linked to the de-regulation of telomerase function and telomere integrity. The activity of helicases plays an important level of regulation of both the telomerase and the organization of the telomere. The proper recognition of the end of chromosomes as a true end leads to chromosome maintenance otherwise it is incorrectly processed as a double-strand break. Our knowledge of the role of motor protein activity at telomeres is of fundamental importance for our understanding of their function as regulators. Thus, the proposed research focuses on the study of their mechanism and it is relevant to the NIH's mission to develop fundamental knowledge that will help understand human diseases.