Control of Genome Stability by Replicative DNA Polymerases

描述（由申请人提供）：本提案解决了生物学中一个长期存在的问题——真核生物中复制性DNA聚合酶（Pol）在保护基因组稳定性和防止复制错误引起的疾病方面的相对贡献。近年来该领域的显著进展使人们认识到，真核生物基因组的精确复制、重组和修复需要多种DNA聚合酶。然而，单个聚合酶的作用还远未被了解。我们设计了一个整体的方法来研究pol的角色。我们将结合体外生化和体内遗传的方法。与现有方法相比，该方法的优点是使用了超诱变碱基类似物、羟胺嘌呤（HAP）和战略性DNA聚合酶突变体。HAP使DNA链特异性错误的检测成为可能，它是基于巴甫洛夫博士多年的研究。具有低碱基选择性和禁用校对外切酶的pol的突变株将使我们能够在体内追踪单个pol。研究的目标将在三个具体目标中实现。首先是通过双突变体分析对DNA Pol相互作用的遗传研究。使用具有不准确Pol a的突变体作为参考，我们期望定义DNA Pol在滞后和领先DNA链上的交易顺序。特异性目标2是确定体外URA3基因中pol及其不准确变体的错误特征。特异性目的3是确定DNA聚合酶在基因组稳定性中的相对作用。在一种方法中，我们将研究在具有不准确Pol a的菌株中位于复制原点附近的报告基因中突变热点的分布。不准确Pol a参与体内复制的起始将导致突变的独特周期性分布与冈崎片段的边界一致。这将首次提供对体内冈崎片段大小的估计。第二种方法是通过强大的统计方法比较同一报告基因中Pols在体外和体内产生的突变谱。在每个体内光谱中发现的DNA聚合酶的突变特征将揭示它们参与前导链和滞后链复制的程度。我们期望Aims 1-3能让我们更好地理解复制分叉处的Pol排列。本研究旨在解决真核生物中不同DNA聚合酶在复制叉中的作用这一面向人类健康的基本生物学问题。由DNA聚合酶错误产生的突变是癌症的主要原因之一。这项工作将建立一个由不准确的DNA聚合酶产生的突变数据库，该数据库可用于寻找人类癌症中相同的突变特征。我们还将发现聚合酶等位基因的组合，它们协同破坏基因组的稳定，并产生患癌症和其他疾病的倾向。公共卫生相关性：该项目旨在估计真核生物复制叉处复制性DNA聚合酶的相对作用，以及DNA聚合酶突变损害其保真度的遗传后果和对人类健康的影响。

DESCRIPTION (provided by applicant): This proposal addresses a long-standing problem in biology - the relative contribution of replicative DNA polymerases (Pol) in eukaryotes in guarding genome stability and protecting from diseases stemming from faults in replication. Remarkable progress in the field in recent years has led to the understanding that multiple DNA polymerases are required for accurate replication, recombination and repair of the eukaryotic genome. However, the roles of individual polymerases are far from being understood. We designed an integral approach to study roles of Pols. We will combine in vitro biochemical and in vivo genetic methods. The advantage over the existing methods is the use of the supermutagenic base analog, hydroxylaminopurine (HAP) and strategic DNA polymerase mutants. HAP makes the detection of DNA strand-specific errors possible and is based on years of Dr. Pavlov's research. Mutator strains possessing Pols with reduced base selectivity and disabled proofreading exonuclease will allow us to track individual Pols in vivo. The goal of the study will be pursued in the three specific aims. First is the genetic study of DNA Pol interplay by double mutant analysis. Using mutants with inaccurate Pol a as a reference, we expect to define the order of DNA Pols transactions on the lagging and leading DNA strands. Specific Aim 2 is the determination of error signatures of Pols and their inaccurate variants in vitro in the URA3 gene. Specific Aim 3 is the determination of the relative roles of DNA polymerases in genome stability. In one approach we will study the distribution of mutation hotspots in the reporter placed near the replication origin in strains with inaccurate Pol a. The involvement of inaccurate Pol a in the initiation of replication in vivo will result in a unique periodic distribution of mutations coinciding with the boundaries of Okazaki fragments. For the first time, this will provide an estimate of the size of Okazaki fragments in vivo. In second approach, the mutation spectra produced by Pols in vitro and generated in vivo in the same reporter gene will be compared by powerful statistical approaches. The mutation signatures of the DNA polymerases found in each of the in vivo spectra will reveal the extent of their participation in the replication of the leading and lagging strands. We expect that Aims 1-3 will lead to a better understanding of the Pol arrangement at the replication fork. The research aims the fundamental human health-oriented biological problem of the roles of different DNA polymerases in the replication fork in eukaryotes. The mutations generated by errors of the DNA polymerases are among the primary causes of cancer. The work will result in a database of mutations generated by inaccurate DNA polymerases, which could be used to find the same mutation signature in human cancers. We will also find a combination of polymerase alleles that synergistically destabilize the genome and create a predisposition to cancer and other diseases. PUBLIC HEALTH RELEVANCE: The project seeks to estimate the relative roles of replicative DNA polymerases at replication fork in eukaryotes and the genetic consequences and impact on human health of mutations in DNA polymerases compromising their fidelity.