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

说明书(申请人提供)：Rev1在DNA聚合酶中是独一无二的，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。我们已经解决的Rev1晶体结构表明了一种优雅的机制，通过这种聚合酶可以促进熟练和无错误的复制，通过大量的氮加成鸟嘌呤，这些鸟嘌呤是由内源性氧化损伤和暴露于大量广泛的DNA破坏性化学和环境致癌物，如丁二烯环氧化物和反苯并[a]芘二醇环氧化物。酵母菌Rev1的这种作用将使用生化、遗传和结构相结合的方法进行检验。在目标1中，与进入的dCTP配对以及与模板G的驱逐和稳定有关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酶和Rev1.DNA二元复合体的晶体结构以及它们与Rev1.DNA.dCTP三元复合体的结构比较，分析Rev1在DNA结合和dNTP结合时发生的构象变化。在目标3中，将进行生物化学研究，以检验这一假设，即Rev1 DNA合成活性的主要作用是通过各种鸟嘌呤的N2加合物促进有效和无错误的复制，这些N2加合物以立体方式影响到小沟，这些N2加合物是由于细胞氧化损伤或暴露于DNA破坏环境致癌物而产生的。另外，作为这一目标的一部分，我们将研究Rev1和延伸聚合酶之间的复合体形成协调这些加合物旁路中核苷酸插入和后续延伸步骤的方法。作为这些生化研究的补充，在目标4中，将确定含有各种N_2鸟嘌呤加合物的DNA的Rev1的晶体结构，以及具有基本损伤的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的各种鸟嘌呤氮加成物促进无错误复制方面的要求。REV1和其他DNA修复蛋白在酵母和人类之间高度保守。Rev1通过鸟嘌呤氮端形成的大量DNA加合物促进复制的熟练和准确能力将通过保持低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损伤进行无错误复制为癌症预防提供了一种重要手段。公共卫生相关性：细胞氧化损伤和暴露于环境污染物所产生的DNA损伤会影响基因组DNA的稳定性和完整性。通过这种病变的无错误复制可以保持低突变率和减少癌症形成的发生率，从而减少它们的不利影响。拟议的研究将检查Rev1 DNA聚合酶在通过DNA损伤促进无错误复制方面的作用。

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.