Role of human DNA polymerase iota in replicative bypass of DNA lesions

描述(由申请人提供)：跨损伤合成(TLS)DNA聚合酶(POL)通过阻止复制叉继续进展的DNA损伤促进复制。尽管对各种酵母和人类TLS POL的结构研究表明，它们可以以高度专业化的方式在TLS中发挥作用，但可用的信息 它们的生物学作用相对较弱；尤其是POL的生物学作用？仍然是最不被理解的。在拟议的研究中，我们将使用遗传、生化和结构相结合的方法来检验Pol？通过损害Watson-Crick(W-C)碱基配对或突出到DNA小沟的DNA损伤促进复制。此外，最重要的是，将进行研究，以检验波尔？与相对的未受损残基相比，与DNA损伤相反的功能以更无错误的方式发挥作用。在目标1中，我们将研究POL？和其他TLS Pol在人体细胞中通过N1-甲基腺嘌呤(1-MeA)促进复制，它损害W-C碱基配对；N3-甲基腺嘌呤(3-DMEA)的去氮衍生物，它突出到DNA小沟；以及1，3-丁二烯(N_2-DG，R-丁二烯单环氧化物)的N_2-DG加合物，它和3-DMEA一样，是轻微的沟槽损伤，但化学上更复杂。1-MEA和3-DMEA是由环境烷基化试剂和内源细胞反应产生的，1，3-丁二烯是一种重要的工业化学品和环境污染物。为了在人类细胞中进行TLS分析，我们将使用两种不同的双链质粒系统，一种是基于SV40起始点的质粒，另一种是基于EBV起始点的质粒，以及Pol？以及其他POL对病变旁路和致突变性的影响将被确定。在目标2中，将进行生化研究以检验POL的熟练程度。以及合成与1-MeA、3-DMEA和N_2-DG R-丁二烯单环氧化物加合物相反的DNA的其他POL。通过稳态动力学分析，确定了POL？以及用于插入与这些病变中的每一个相对的核苷酸(NT)并用于从插入的NT延伸的其他POL。在目标3中，波兰人的结构？在与1-MeA、3-DMEA、N_2-DG-R-丁二烯单环氧化物的三元络合物中，还将测定一个(6-4)TT光解产物，以揭示Pol？能够以一种主要无错误的方式与这些DNA损伤相对发挥作用。将在人类细胞中进行生化研究和TLS研究，以检查残基突变的影响，这些突变有助于稳定与病变部位相反的正确进入的NT。拟议的研究与癌症生物学和癌症病因学高度相关，因为它们将揭示POL？与其他TLS POL一起，是否促进TLS的主要无错误模式，而不是多样化的DNA加合物阵列。TLS的无错误模式将与Pol的角色保持一致？在抑制因接触环境和化学致癌物而导致的致癌方面。

DESCRIPTION (provided by applicant): Translesion synthesis (TLS) DNA polymerases (Pols) promote replication through DNA lesions which block the continued progression of the replication fork. Although structural studies with the various yeast and human TLS Pols have indicated that they could function in TLS in highly specialized ways, the information available for their biological roles has been relatively meager; in particular, the biological role of Pol? has remained the least understood. In the proposed studies, we will use a combined genetic, biochemical, and structural approach to test the hypothesis that Pol? makes an important contribution to promoting replication through DNA lesions which impair Watson-Crick (W-C) base pairing or which protrude into the DNA minor groove. Furthermore, and most importantly, studies will be done to test the hypothesis that Pol? functions opposite DNA lesions in a much more error-free manner than opposite undamaged residues. In Aim 1, we will examine the roles of Pol? and other TLS Pols in human cells in promoting replication through N1-methyl adenine (1-MeA), which impairs W-C base pairing; a deaza derivative of N3-methyl adenine (3-dMeA), which protrudes into the DNA minor groove; and an N2-dG adduct of 1,3-butadiene (N2-dG, R-butadiene monoepoxide), which like 3-dMeA, is a minor groove lesion but chemically more complex. 1-MeA and 3-dMeA are generated from exposure to environmental alkylating agents and from endogenous cellular reactions, and 1,3-butadiene is an important industrial chemical and an environmental pollutant. For TLS analysis in human cells, we will utilize two different duplex plasmid systems, an SV40 origin-based plasmid and an EBV origin-based plasmid, and the relative contributions of Pol? and of other Pols to lesion bypass and to mutagenicity will be determined. In Aim 2, biochemical studies will be done to examine the proficiency of Pol? and of other Pols in synthesizing DNA opposite the 1-MeA, 3-dMeA, and N2-dG R-butadiene monoepoxide adducts. By steady-state kinetic analyses, we will determine the catalytic efficiency and fidelity of Pol? and of other Pols for inserting a nucleotide (nt) opposite each of these lesions and for extending from the inserted nt. In Aim 3, structures of Pol? in ternary complex with the 1-MeA, 3-dMeA, N2-dG R-butadiene monoepoxide, and also a (6-4) TT photoproduct will be determined to uncover the bases of Pol? ability to function opposite these DNA lesions in a predominantly error-free manner. Biochemical studies, and TLS studies in human cells, will be carried out to examine the effects of mutations in residues that help stabilize the correct incoming nt opposite the lesion site. The proposed studies are highly relevant for cancer biology and cancer etiology as they will reveal whether Pol?, in conjunction with other TLS Pols, promotes a predominantly error-free mode of TLS opposite a diverse array of DNA adducts. An error-free mode of TLS would be in keeping with a role for Pol? in suppression of carcinogenesis that would otherwise result from exposure to environmental and chemical carcinogens.