Role of Rev1 in error-free replication of DNA damage and in mutation prevention

Rev1 在 DNA 损伤的无差错复制和突变预防中的作用

• 批准号: 7462040
• 负责人: SATYA PRAKASH
• 金额: $ 42.86万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7462040
• 关键词: 2' -deoxycytidine 5' -triphosphate; Acrolein; Affect; Aldehydes; Amino Acids; Apoenzymes; Arginine; Benzo(a)pyrene; Binding; Biochemical; Biochemical Genetics; Butadiene; Bypass; Cancer Biology; Cells; Complement; Complex; DNA; DNA Adducts; DNA Binding; DNA Damage; DNA Replication Damage; DNA biosynthesis; DNA chemical synthesis; DNA lesion; DNA repair protein; DNA-Directed DNA Polymerase; Environmental Carcinogens; Environmental Pollutants; Epoxy Compounds; Etiology; Eukaryota; Eukaryotic Cell; Exposure to; Genetic; Genome Stability; Genomics; Glycol; Goals; Guanine; Helix (Snails); Human; Hydrogen Bonding; Incidence; Lesion; Lipid Peroxidation; Malignant Neoplasms; Minor Groove; Mutate; Mutation; Nucleotides; Numbers; Plasmids; Polymerase; Prevention; Proteins; Public Health; Rate; Reaction; Role; Site; Specificity; Structure; Testing; Yeasts; adduct; base; cancer prevention; carcinogenesis; ear helix; environmental chemical; in vivo; insight; nucleotide analog; synthetic construct

DESCRIPTION (provided by applicant): Rev1 is unique among DNA polymerases in that the protein itself rather than the DNA template determines the specificity for both the templating and the incoming nucleotide. The Rev1 crystal structure that we have solved suggests an elegant mechanism by which this polymerase could promote proficient and error-free replication through a large variety of N2-adducted guanines that result from endogenous oxidative damage and from exposure to a number of widespread DNA damaging chemical and environmental carcinogens such as butadiene epoxides and anti-benzo[a]pyrene diol epoxides. Such a role for yeast Rev1 will be examined using a combined biochemical, genetic, and structural approach. In Aim 1, key amino acid residues involved in the pairing with the incoming dCTP and in the eviction and stabilization of templating G will be mutated and their effects on nucleotide incorporation specificity and catalytic efficiency determined. In Aim 2, the conformational changes that occur in Rev1 upon DNA binding and upon dNTP binding will be analyzed through crystal structures of the Rev1 apoenzyme and Rev1.DNA binary complex and their comparison to the structure of Rev1.DNA.dCTP ternary complex. In Aim 3, biochemical studies will be undertaken to test the hypothesis that a major role of the Rev1 DNA synthetic activity is to promote efficient and error-free replication through various N2adducts of guanine that sterically impinge upon the minor groove, and which result from cellular oxidative damage or from exposure to DNA damaging environmental carcinogens. Also as part of this aim, we will examine the means by which complex formation between Rev1 and the extender polymerase coordinates the nucleotide insertion and the subsequent extension steps in the bypass of these adducts. As a complement to these biochemical studies, in Aim 4, crystal structures of Rev1 with DNAs containing a variety of N2 guanine adducts will be determined, as well as the structure of Rev1 with an abasic lesion. In Aim 5, genetic studies will be done to establish the requirement of the Rev1 DNA synthetic activity in promoting error-free replication through the various N2-adducts of guanine in yeast cells. Rev1 as well as the other DNA repair proteins are highly conserved between yeast and humans. The proficient and accurate ability of Rev1 for promoting replication through the large variety of DNA adducts that form at the N2 of guanine will have a major impact on genome stability by keeping the rate of mutations low, reducing thereby the incidence of carcinogenesis in humans. The results of this study are highly relevant for cancer biology and etiology, as error-free replication through DNA lesions provides for an important means of cancer prevention. PUBLIC HEALTH RELEVANCE: DNA lesions generated from cellular oxidative damage and from exposure to environmental pollutants affect the stability and integrity of genomic DNA. Error-free replication through such lesions reduces their adverse impact by keeping the rate of mutations low and by reducing the incidence of cancer formation. The proposed studies will examine the role of Rev1 DNA polymerase in promoting error-free replication through DNA lesions.

描述（由申请人提供）：Rev1 在 DNA 聚合酶中是独特的，因为蛋白质本身而不是 DNA 模板决定了模板和引入核苷酸的特异性。我们解决的Rev1晶体结构表明了一种优雅的机制，通过这种机制，这种聚合酶可以通过多种N2加合鸟嘌呤促进熟练且无差错的复制，这些鸟嘌呤是由内源性氧化损伤和暴露于许多广泛存在的DNA损伤化学物质引起的以及环境致癌物，例如丁二烯环氧化物和抗苯并[a]芘二醇环氧化物。将使用生化、遗传和结构相结合的方法来检查酵母 Rev1 的这种作用。在目标 1 中，参与与传入 dCTP 配对以及模板 G 的驱逐和稳定的关键氨基酸残基将发生突变，并确定它们对核苷酸掺入特异性和催化效率的影响。在目标 2 中，将通过 Rev1 脱辅基酶和 Rev1.DNA 二元复合物的晶体结构以及它们与 Rev1.DNA.dCTP 三元复合物的结构的比较来分析 Rev1 在 DNA 结合和 dNTP 结合时发生的构象变化。在目标 3 中，将进行生化研究来检验这一假设，即 Rev1 DNA 合成活性的主要作用是通过鸟嘌呤的各种 N2 加合物促进有效且无差错的复制，这些鸟嘌呤 N2 加合物空间撞击小沟，并且由细胞产生氧化损伤或暴露于 DNA 损伤性环境致癌物质。同样作为该目标的一部分，我们将检查 Rev1 和延伸聚合酶之间的复合物形成协调核苷酸插入以及绕过这些加合物的后续延伸步骤的方式。作为这些生化研究的补充，在目标 4 中，将确定 Rev1 与含有各种 N2 鸟嘌呤加合物的 DNA 的晶体结构，以及具有脱碱基损伤的 Rev1 的结构。在目标 5 中，将进行遗传学研究，以确定 Rev1 DNA 合成活性在通过酵母细胞中鸟嘌呤的各种 N2 加合物促进无差错复制方面的要求。 Rev1 以及其他 DNA 修复蛋白在酵母和人类之间高度保守。 Rev1 通过在鸟嘌呤 N2 处形成的多种 DNA 加合物促进复制的熟练而准确的能力将通过保持较低的突变率对基因组稳定性产生重大影响，从而降低人类致癌的发生率。这项研究的结果与癌症生物学和病因学高度相关，因为通过 DNA 损伤进行的无差错复制提供了预防癌症的重要手段。公共卫生相关性：细胞氧化损伤和环境污染物暴露产生的 DNA 损伤会影响基因组 DNA 的稳定性和完整性。通过此类病变的无差错复制可保持较低的突变率并降低癌症形成的发生率，从而减少其不利影响。拟议的研究将检验 Rev1 DNA 聚合酶在通过 DNA 损伤促进无差错复制中的作用。