Creation and Repair of Postreplicative DNA Gaps

项目概要/摘要 当复制叉遇到模板链中的DNA损伤时，复制让位于DNA修复 和重组。这些接触定义了DNA代谢中的一个界面， 定义基因组不稳定性的DNA重排、重复扩增和诱变。这最终是 表现在真核生物的肿瘤进化和抗生素耐药性的发展， 细菌的致病性最近对分叉处发生的事件的调查在很大程度上忽略了一个 修复的重要基因组场所--复制后缺口中分叉后留下的损伤。存在这些 50多年来，人们一直认识到存在差距，但进展受到方法的限制， 不足以适当地探讨其普遍的重要性和修复。这些间隙是 通过跨损伤DNA聚合酶的DNA合成、重组DNA修复和复制模板 开关，所有的过程都与基因组的不稳定性有关。 这一建议框架的多学科努力，探讨如何产生后复制差距，多久 它们是形成的，什么情况触发了形成，它们内部发生了什么，以及各种路径是如何形成的。 优先考虑并管理缺口修复。这项工作是理解三个方面的进步的产物。 神秘的蛋白活性，RarA，Uup和RadD。RarA，一种AAA+ ATP酶，在复制体中起作用， 在滞后链上产生复制后的缺口，是一个关键。 为了解决这一问题，我们汇集了生物化学、遗传学、分子生物学和生物医学领域的世界级专业知识， 生物学和生物物理学。我们将开发新的方法，包括新的单分子方法， 检测和量化缺口，并表征作用于缺口的蛋白质。在我们的驱动下， 机械问题，这些方法将广泛受益于基因组维护的研究。 这五个具体目标构成了对这一问题的系统攻击。RarA蛋白的作用机制是 目标1的焦点。目的2提供了第一个努力，以量化间隙形成在体内，并确定因素， 触发间隙形成。目的3探讨Uup和RadD这两种抑制模板的酶的功能 在间隙中切换。目标4探讨了如何组织和管理缺口修复的各种途径。的 一项提案完成了目标5，探索跨损伤DNA聚合酶在间隙内的作用，这是一个关键 基因组诱变的来源。

PROJECT SUMMARY/ABSTRACT When a replication fork encounters a DNA lesion in a template strand, replication gives way to DNA repair and recombination. These encounters define an interface in DNA metabolism that gives rise to much of the DNA rearrangement, repeat expansion, and mutagenesis that defines genome instability. This is ultimately manifested in tumor evolution in eukaryotes and the development of antibiotic resistance and increased pathogenicity in bacteria. Recent investigation of events that occur at the fork have largely overlooked an important genomic venue for repair – lesions left behind the fork in postreplicative gaps. The existence of these gaps has been appreciated for over 5 decades, but progress has been limited by methodology that has been inadequate to properly explore their general importance and repair. These gaps are primary substrates for DNA synthesis by translesion DNA polymerases, recombinational DNA repair, and replicational template switching, all processes linked with genomic instability. This proposal frames a multidisciplinary effort to explore how postreplicative gaps are generated, how often they are formed, what circumstances trigger formation, what occurs within them, and how the various paths of gap repair are prioritized and governed. The work is an outgrowth of advances in understanding three enigmatic protein activities, RarA, Uup, and RadD. RarA, an AAA+ ATPase that functions at the replisome to generate postreplicative gaps on the lagging strand, is one key. To tackle this problem, we bring together world-class expertise in biochemistry, genetics, molecular biology, and biophysics. We will develop new methods, including novel single-molecule approaches towards detecting and quantifying gaps, and characterizing the proteins acting on them. While driven by our mechanistic questions, these methods will broadly benefit research in genomic maintenance. The five specific aims constitute a systematic attack on the problem. The mechanism of RarA protein is the focus of Aim 1. Aim 2 provides the first effort to quantify gap formation in vivo and determine the factors that trigger gap formation. Aim 3 explores the functions of Uup and RadD, two enzymes that suppress template switching within gaps. Aim 4 explores how various pathways of gap repair are organized and governed. The proposal is completed with Aim 5, an exploration of translesion DNA polymerases acting within gaps, a key source of genomic mutagenesis.