Genetic control of replication through DNA lesions in humans, and carcinogenesis

描述(由申请人提供)：跨损伤合成(TLS)DNA聚合酶(POL)通过促进DNA损伤的复制，帮助确保复制分叉的持续进展。在拟议的研究中，我们将确定一些人TLS Pol在通过环境污染物和致癌物以及细胞氧化损伤引起的各种DNA损伤促进复制方面的作用。特别是，我们将测试这一假设，即通过人类细胞中的DNA损伤进行复制通过两种不同的模式发生，其中POLS、？、？、K、Rev1和？调解以无错误为主的TLS，并根据DNA损伤以高度专业化的方式发挥作用，而Pol？以更普遍和更容易出错的方式执行病变旁路。进一步，我们将检验假设，更普遍和更容易出错的作用？它的产生是因为它能够通过“蛋白质模板”导向的机制插入一个嘌呤核苷酸(NT)，优先是A，与DNA损伤相反。为了阐明人类通过DNA损伤进行无错误和突变复制的遗传学基础，我们将开展以下研究。在目标1中，我们将研究不同的TLS POL对人类和小鼠细胞无错误与突变病变搭桥的贡献。要研究的损害包括：(6-4)紫外线照射产生的光产物，DNA中鸟嘌呤自由基攻击产生的7，8-二氢-8-氧鸟苷(8-oxoG)，腺嘌呤与细胞氧化损伤和接触化学致癌物引起的脂质过氧化产物相互作用产生的1，N6-亚乙基脱氧腺苷(EDA)，1，N-丙基-2‘-脱氧鸟苷(PDG)，它是一种由脂质过氧化产生的丙烯醛的闭环形式，也是一种普遍存在的环境污染物，以及环境致癌物质(反苯并[a]expoxide)的N2-DG加合物(PDBE)。在目标2中，将进行生化研究以检验POL的熟练程度。在合成DNA对(6-4)TT光产物中，8-oxoG、eda、pdG和n2-dG BPDE。通过稳态动力学分析，确定了POL？为了在DNA损伤的对面插入核苷酸并进行随后的延伸反应，将进行生化研究以验证Pol？通过蛋白质模板导向的机制插入与DNA损伤相反的嘌呤NT。我们在这里提出的遗传和生化研究对于描述TLS POLS在促进复制过程中无错误和突变损伤旁路中的作用，以及为全面了解环境和细胞DNA损伤剂在人类细胞中诱导突变和致癌的遗传基础具有重要意义。我们对Pol？、？、k、Rev1和？提出的TLS的主要无错误模式的建议。会预测这些POL在癌症抑制中的作用，而POL对TLS的突变模式？将预测这种POL在增强基因组不稳定性和致癌方面的作用。 与公共卫生相关：DNA损伤是由于细胞氧化损伤以及接触化学和环境致癌物而在人类细胞中产生的。在人类细胞复制过程中，确定各种DNA聚合酶在促进无错误和诱变损伤旁路中的作用，对于全面了解环境和细胞DNA损伤剂诱导突变和致癌的遗传基础是重要的。

DESCRIPTION (provided by applicant): Translesion synthesis (TLS) DNA polymerases (Pols) help ensure the continued progression of the replication fork by promoting replication through DNA lesions. In the proposed studies, we will determine the roles of a number of human TLS Pols in promoting replication through a variety of DNA lesions induced by environmental pollutants and carcinogens and by cellular oxidative damage. In particular, we will test the hypothesis that replication through DNA lesions in human cells occurs via two distinct modes in which Pols, ?, ?, k, Rev1, and ? mediate predominantly error-free TLS and act in a highly specialized manner dependent upon the DNA lesion, whereas Pol? performs lesion bypass in a more generalized and error-prone manner. Further, we will test the hypothesis that the more generalized and error-prone role of Pol? emanates from its ability to insert a purine nucleotide (nt), preferentially an A, opposite DNA lesions via a "protein-template"-directed mechanism. To elucidate the genetic bases of error-free and mutagenic replication through DNA lesions in humans, we will carry out the following studies. In Aim 1, we will examine the contributions of various TLS Pols to error-free vs. mutagenic lesion bypass in human and mouse cells. The lesions to be studied include (6- 4) photoproduct induced by UV irradiation, 7,8-dihydro-8-oxogunaine (8-oxoG) generated from free-radical attack on guanine in DNA, 1,N6-ethenodeoxyadenosine (edA) generated from interaction of adenine with products of lipid peroxidation resulting from cellular oxidative damage and from exposure to chemical carcinogens, 1,N2-propano-2'-deoxyguanosine (PdG), a ring-closed form of acrolein generated from lipid peroxidation and which also is a ubiquitous environmental pollutant, and N2-dG adduct of the environmental carcinogen (+) anti-benzo[a]pyrene diol expoxide (BPDE). In Aim 2, biochemical studies will be done to examine the proficiency of Pol? in synthesizing DNA opposite (6-4) TT photoproduct, 8-oxoG, edA, PdG, and N2-dG BPDE. By steady-state kinetic analyses we will determine the catalytic efficiency of Pol? for inserting nucleotides opposite the DNA lesion and for carrying out the subsequent extension reaction, and biochemical studies will be done to test the hypothesis that Pol? inserts a purine nt opposite DNA lesions via a protein- template-directed mechanism. The genetic and biochemical studies we propose here are highly relevant for delineating the roles of TLS Pols in promoting error-free vs. mutagenic lesion bypass during replication, and for providing a comprehensive understanding of the genetic bases of mutagenesis and carcinogenesis induced by environmental and cellular DNA damaging agents in human cells. Our proposal for a predominantly error-free mode of TLS by Pols ?, ?, k, Rev1, and ? would predict a role for these Pols in cancer suppression, whereas a mutagenic mode of TLS by Pol? would predict a role for this Pol in enhancing genomic instability and carcinogenesis. PUBLIC HEALTH RELEVANCE: DNA lesions are generated in human cells from cellular oxidative damage and from exposure to chemical and environmental carcinogens. The determination of roles of various DNA polymerases in promoting error-free vs. mutagenic lesion bypass during replication in human cells is important for providing a comprehensive understanding of the genetic bases of mutagenesis and carcinogenesis induced by environmental and cellular DNA damaging agents.