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聚合酶之间是唯一的，因为蛋白质本身而不是DNA模板决定了模板和输入核苷酸的特异性。我们已解决的Rev1晶体结构提出了一种优雅的机制，该聚合酶可以通过该机制通过多种N2添加的N2添加鸟嘌呤来促进熟练且无错误的复制，这些鸟嘌呤是由内源性氧化损伤以及暴露于多种损害DNA的化学化学物质造成的。和环境致癌物，例如丁二烯环氧化物和抗苯并[A] pyrene二醇环氧化物。将使用合并的生化，遗传和结构方法来检查酵母Rev1的这种作用。在AIM 1中，将突变与传入的DCTP以及模板G的驱逐和稳定中涉及的关键氨基酸残基被突变，并且它们对确定的核苷酸掺入特异性和催化效率的影响。在AIM 2中，将通过Rev1 apoenzyme和Rev1.DNA二元复合物的晶体结构分析REV1和DNTP结合后Rev1中发生的构象变化及其与Rev1.dna.dna.dna.dctp ternary综合体的结构的比较。在AIM 3中，将进行生化研究，以检验以下假设：Rev1 DNA合成活性的主要作用是通过鸟嘌呤的各种N2ADDUCTS促进有效且无错误的复制，而鸟嘌呤在空间上造成了次要的凹槽，并且由细胞造成。氧化损伤或暴露于DNA破坏环境致癌物。同样，作为此目标的一部分，我们将研究Rev1和扩展聚合酶之间的复杂形成的手段，可以协调核苷酸插入以及这些加合物旁路的后续扩展步骤。作为对这些生化研究的补充，在AIM 4中，将确定Rev1的Rev1的晶体结构，其中包含多种N2鸟嘌呤加合物的DNA，以及带有abasic病变的Rev1结构。在AIM 5中，将进行遗传研究，以确定Rev1 DNA合成活性的要求，以通过酵母细胞中鸟嘌呤的各种N2-添加促进无错误复制。 Rev1以及其他DNA修复蛋白在酵母和人之间高度保守。 Rev1通过在鸟嘌呤N2形成的各种DNA加合物中促进复制的能力促进复制，这将通过保持较低的突变速率来对基因组稳定产生重大影响，从而降低人类癌变的发生率。这项研究的结果与癌症生物学和病因高度相关，因为通过DNA病变的无错误复制提供了预防癌症的重要手段。公共卫生相关性：由细胞氧化损伤和暴露于环境污染物的DNA病变会影响基因组DNA的稳定性和完整性。通过这种病变通过这种病变的无误复制可通过保持较低的突变速率和降低癌症形成的发生率来减少其不良影响。拟议的研究将研究Rev1 DNA聚合酶在通过DNA病变促进无错误复制中的作用。