HELICASE ACTIVITY AND ITS ROLE IN TELOMERE AND TELOMERASE REGULATION

描述(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真正的末端还是错误地被识别为双链断裂的基本步骤。近年来，发动机蛋白(如解旋酶)在端粒调节中的作用已变得明显。即使在酵母这一研究最深入的模型系统中，解旋酶在端粒调节中的作用也不完全清楚。例如，目前尚不清楚这些马达蛋白的主要功能是来自它们的解旋酶活性，从而它们解开双链核酸的能力，还是它们沿着DNA移位和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置换来影响端粒的功能，端粒酶是一种专门的逆转录酶，负责延伸端粒的富含G的链。第二个解旋酶Rrm3与端粒功能有关。RRM3促进了包括端粒在内的1000个染色体部位的复制分叉进程。Rrm3在端粒复制分叉过程中的一个可能功能是结合到端粒双链区域的Rap1的位移。了解Pif1和Rrm3的内在生化特性以及它们是如何被利用/改变来实现其功能的，对于阐明这些马达蛋白如何参与端粒调节是必要的。在特定的目标1中，我们将：a)研究Pif1-DNA相互作用的核苷酸辅助因子对变构的调节。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对端粒酶的抑制是否源于其解旋酶活性或其取代端粒酶复合体亚单位的能力。在特定目标2中，我们将：a)检验我们的假设，即RRM3的ATPase和解旋酶活性受其N-末端区域内的序列/结构域调控。B)检验以下假设，即增殖细胞核抗原与RRM3结合可解除其N-末端区域的调节功能。C)测试与增殖细胞核抗原相互作用导致活性RRM3能够从端粒上取代RAP1的假设。这些研究将使我们能够确定Pif1和Rrm3是如何发挥作用的，并将为这些酶如何参与端粒/端粒酶调控提供独特的见解。我们希望确定Pif1的寡聚化是否导致其功能分离，从而确定其在端粒上的特定作用。此外，我们希望确定如何调节RRM3的活性，以允许移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。 公共卫生相关性：拟议的研究与公共健康相关，因为染色体不稳定、肿瘤发生和细胞衰老与端粒酶功能和端粒完整性的去调节有关。解旋酶的活性在端粒酶和端粒组织的调节中起着重要的作用。正确地将染色体末端识别为真正的末端会导致染色体的维持，否则它会被错误地处理为双链断裂。我们对端粒上运动蛋白活性的了解对于我们理解它们作为调节者的功能是至关重要的。因此，拟议的研究重点是研究它们的机制，这与美国国立卫生研究院发展有助于理解人类疾病的基础知识的使命相关。

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.