Control of Genome Stability by Replicative DNA Polymerases

通过复制 DNA 聚合酶控制基因组稳定性

• 批准号: 7806946
• 负责人: YOURI I PAVLOV
• 金额: $ 1.11万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7806946
• 关键词: 6-N-hydroxylaminopurine deoxynucleoside triphosphate; Address; Affect; Alleles; Biochemical; Biological; Biological Models; Biology; Bypass; Cancer Etiology; Cells; Chromosomes; Complex; DNA; DNA Damage; DNA Sequence; DNA biosynthesis; DNA-Directed DNA Polymerase; Data; Databases; Defect; Detection; Diploid Cells; Diploidy; Disabled Persons; Disease; Enzymes; Eukaryota; Event; Exonuclease; Funding; Future; Generations; Genes; Genetic; Genetic Materials; Genome; Genome Stability; Genomic Instability; Goals; Haploidy; Health; Human; In Vitro; Individual; Induced Mutation; Laboratories; LacZ Genes; Lead; Maintenance; Malignant Neoplasms; Methods; Mismatch Repair; Monitor; Mutagenesis; Mutation; Mutation Spectra; Noise; Okazaki fragments; Polymerase; Positioning Attribute; Predisposition; Prevention; Process; Proteins; Reagent; Relative (related person); Replication Initiation; Replication Origin; Reporter; Reporter Genes; Research; Role; Signal Transduction; Site; Source; Specificity; Spectrum Analysis; Statistical Methods; System; Testing; Thinking; Time; Uncertainty; Variant; Work; Yeasts; analog; base; cancer risk; comparative; design; human disease; in vivo; innovation; mutant; novel; prevent; protein function; public health relevance; recombinational repair; repaired; replicase; research study; stem; tripolyphosphate

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.

描述（由申请人提供）：该提案解决了生物学中长期存在的问题 - 真核生物中复制 DNA 聚合酶 (Pol) 在保护基因组稳定性和防止复制缺陷引起的疾病方面的相对贡献。近年来该领域取得的显着进展使人们认识到真核基因组的精确复制、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。我们设计了一种整体方法来研究 Pols 的角色。我们将结合体外生化和体内遗传方法。与现有方法相比的优点是使用超诱变碱基类似物、羟氨基嘌呤 (HAP) 和策略性 DNA 聚合酶突变体。 HAP 使 DNA 链特异性错误的检测成为可能，并且基于 Pavlov 博士多年的研究。具有降低碱基选择性和禁用校对核酸外切酶的 Pol 的突变菌株将使我们能够在体内追踪单个 Pol。研究的目标将围绕三个具体目标来实现。首先是通过双突变体分析对 DNA Pol 相互作用进行遗传学研究。使用 Pol a 不准确的突变体作为参考，我们期望定义落后和领先 DNA 链上 DNA Pols 事务的顺序。具体目标 2 是在体外确定 URA3 基因中 Pols 及其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 Pol a 不准确的菌株中位于复制起点附近的报告基因中突变热点的分布。不准确的 Pol a 参与体内复制的启动将导致突变的独特周期性分布，与冈崎片段的边界一致。这将首次提供体内冈崎碎片大小的估计。在第二种方法中，将通过强大的统计方法对同一个报告基因中 Pols 在体外产生的突变谱和体内产生的突变谱进行比较。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。我们预计目标 1-3 将有助于更好地理解复制叉上的 Pol 排列。该研究旨在解决真核生物复制叉中不同DNA聚合酶的作用这一以人类健康为导向的基本生物学问题。 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，可用于在人类癌症中寻找相同的突变特征。我们还将发现聚合酶等位基因的组合，它们会协同破坏基因组的稳定性并导致癌症和其他疾病的易感性。公共健康相关性：该项目旨在估计真核生物复制叉中复制 DNA 聚合酶的相对作用，以及损害其保真度的 DNA 聚合酶突变的遗传后果和对人类健康的影响。