Control of Genome Stability by Replicative DNA Polymerases

描述(申请人提供)：这项建议解决了生物学中的一个长期存在的问题--真核生物中复制DNA聚合酶(POL)在保护基因组稳定和防止因复制错误而引起的疾病方面的相对贡献。近年来该领域的显著进展导致人们认识到，真核基因组的准确复制、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。我们设计了一个完整的方法来研究POL的作用。我们将结合体外生化和体内遗传方法。与现有方法相比，该方法的优点是使用了超致突变碱基类似物、羟氨基嘌呤(HAP)和DNA聚合酶策略突变体。HAP基于巴甫洛夫博士多年的研究，使检测DNA链特定错误成为可能。突变菌株具有碱基选择性降低和校对核酸外切酶失效的POL，这将使我们能够在体内跟踪单个POL。这项研究的目标将在三个具体目标中进行。首先是利用双突变分析对DNA Pol互作的遗传学研究。使用带有不准确POLA的突变体作为参考，我们希望定义滞后和领先DNA链上DNA POLS交易的顺序。特异性目标2是在体外确定POL及其在URA3基因中的不准确变体的错误特征。具体目标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，我们将研究位于复制起点附近的报告中突变热点的分布。不准确的Pola参与体内复制的启动将导致与Okazaki片段边界一致的独特的周期性突变分布。这将是第一次提供对体内冈崎碎片大小的估计。在第二种方法中，POLS在体外产生的突变谱与在体内产生的同一报告基因的突变谱将用强大的统计方法进行比较。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领先和滞后链复制的程度。我们预计，目标1-3将有助于更好地理解复制分叉上的POL安排。这项研究旨在解决与人类健康相关的生物学问题，即不同的DNA聚合酶在真核生物复制分叉中的作用。DNA聚合酶的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，可以用来在人类癌症中找到相同的突变特征。我们还将发现一种聚合酶等位基因的组合，它们协同作用破坏基因组的稳定，并产生癌症和其他疾病的易感性。公共卫生相关性：该项目旨在评估真核生物复制分叉处复制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.