Creation and Repair of Postreplicative DNA Gaps

项目摘要/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。这些相遇定义了DNA新陈代谢中的一个界面，该界面导致了许多 DNA重排、重复扩张和突变定义了基因组的不稳定性。这最终是 表现为真核生物肿瘤的进化和抗药性的发展并增加 细菌中的致病性。最近对叉路口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--断裂后缝隙中分叉后留下的损伤。这些东西的存在 差距已经被认识到50多年了，但进展受到一直以来的方法的限制 不足以适当地探讨它们的一般重要性和修复。这些间隙是主要的底物 跨损伤DNA聚合酶、重组DNA修复和复制模板的DNA合成 切换，所有的过程都与基因组的不稳定有关。 这项提案构建了一项多学科的努力，以探索复制后差距是如何产生的，多长时间一次 它们是形成的，什么环境触发形成，它们内部发生了什么，以及它们的各种路径是如何形成的 差距修复是优先考虑和管理的。这项工作是在理解三个方面取得进展的结果 谜团蛋白活性、RARA、UUP和RADD。RARA，一种在复制体上起作用的AAA ATPase 在滞后的链上产生复制后的间隙，是一个关键。 为了解决这个问题，我们汇集了生物化学、遗传学、分子生物学等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来 检测和量化缺口，并表征作用于缺口上的蛋白质。虽然由我们的 机械问题，这些方法将广泛有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的重点。目标2提供了第一次努力来量化体内缺口的形成并确定 触发缺口形成。目的3探索UUP和RADD这两种抑制模板的酶的功能 在空隙中切换。目标4探讨如何组织和管理缺口修复的各种途径。这个 完成提案的是目标5，探索在空隙中作用的跨病变DNA聚合酶，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.