Functions of DNA helicases at hard-to-replicate sites and telomere regulation

摘要 这项提案的总体目标是回答有关DNA的两个基本问题 新陈代谢：1)DNA复制是如何在难以复制的位置及时有效地进行的； 2)大的核蛋白复合体是如何组成端粒、末端线形的 染色体，被组装和调节。第一个问题出现在我们正在进行的 保守的解旋酶Pif1家族的机制研究。我们的工作突出了 这些解旋酶在促进DNA复制中的基础作用 DNA结构和蛋白质屏障。展望未来，我们寻求解决以下问题：什么 解旋酶活性与聚合酶合成DNA的机制；这种协同作用是如何实现的 导致消除在细胞核或在细胞核中遇到的蛋白质障碍所带来的障碍 线粒体；是否清除障碍是这类解旋酶的一般特性。这个 第二个问题是新的，是基于我们最近发现的酵母和 人端粒DNA结合蛋白；即这些蛋白与 端粒DNA以不同的方式重复，以促进DNA缩合。我们的目标是 测试这些特性是否在建立端粒长度区分中起作用 可访问性。我们通过使用生化重组系统和 采用集成整体生化和生物物理的多管齐下的方法 技术与单分子方法，从而提供了无与伦比的访问行为的方式 单个分子。

Abstract The overall objective of this proposal is to answer two fundamental questions on DNA metabolism: 1) how DNA replication progresses timely and efficiently at hard-to-replicate sites; and 2) how the large nucleoprotein complexes that comprise telomeres, the end of linear chromosomes, are assembled and regulated. The first question emerges from our ongoing mechanistic studies on the conserved Pif1-family of helicases. Our work highlights the fundamental role that these helicases have in facilitating DNA replication at both secondary DNA structures and protein barriers. Going forward, we seek to address the following: what mechanisms couple the helicase activity to DNA synthesis by polymerases; how this synergy leads to removal of obstacles imparted by protein barriers encountered in the nucleus or in mitochondria; whether removal of obstacles is a general property of this class of helicases. The second question is novel and is based on our recent finding of new properties of both yeast and human telomere DNA binding proteins; namely, the ability of these proteins to interact with telomeric DNA repeats in alternative modes and to facilitate DNA condensation. Our goal is to test whether these properties play a role in establishing telomere length discrimination and accessibility. We tackle these problems by using biochemically reconstituted systems and employing a multipronged approach that integrates ensemble biochemical and biophysical techniques with single-molecule methods, thus providing unparalleled access to the behavior of individual molecules.