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关于DNA的两个基本问题 代谢：1）DNA复制如何在难以复制的位点及时有效地进行; 以及2）如何大的核蛋白复合物，包括端粒，末端的线性 染色体，组装和调节。第一个问题来自我们正在进行的 解旋酶Pif 1保守家族的机制研究。我们的工作突出了 这些解旋酶在促进DNA复制中的基本作用， DNA结构和蛋白质屏障。展望未来，我们寻求解决以下问题： 解旋酶活性与聚合酶的DNA合成机制;这种协同作用是如何产生的？ 导致细胞核中或细胞内遇到的蛋白质屏障所造成的障碍被清除， 线粒体;是否清除障碍是这类解旋酶的一般性质。的 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，这些蛋白质与人端粒DNA结合蛋白相互作用的能力。 端粒DNA重复的交替模式，并促进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来解决这些问题， 采用多管齐下的方法， 技术与单分子的方法，从而提供了无与伦比的访问的行为， 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.