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

描述（由申请人提供）：翻译合成（TLS） DNA聚合酶（pol）通过促进DNA损伤的复制来帮助确保复制叉的持续进展。在拟议的研究中，我们将确定一些人类TLS pol在通过环境污染物和致癌物以及细胞氧化损伤诱导的各种DNA损伤促进复制中的作用。特别是，我们将测试假设，通过DNA损伤在人类细胞中复制通过两种不同的模式发生，其中Pols， ？,吗?， k, Rev1，和？主要介导无错误的TLS，并以高度专业化的方式依赖于DNA损伤，而Pol？以更广泛和更容易出错的方式进行病变旁路。此外，我们将检验一个假设，即Pol？源于其通过“蛋白质模板”导向机制插入嘌呤核苷酸（nt）的能力，优先是a，相反的DNA损伤。为了阐明通过人类DNA损伤进行无差错和诱变复制的遗传基础，我们将开展以下研究。在Aim 1中，我们将研究各种TLS pol对人类和小鼠细胞中无差错和诱变病变旁路的贡献。待研究的病变包括紫外线照射诱导的（6- 4）光产物，DNA中鸟嘌呤自由基攻击产生的7,8-二氢-8-氧古碱（8-oxoG），腺嘌呤与细胞氧化损伤和暴露于化学致癌物引起的脂质过氧化产物相互作用产生的1，n6 -乙基脱氧腺苷（edA）， 1，n2 -丙基-2'-脱氧鸟嘌呤（PdG），丙烯醛是一种由脂质过氧化生成的环状封闭形式，也是一种普遍存在的环境污染物，以及环境致癌物（+）抗苯并[a]芘二醇暴露物（BPDE）的N2-dG加合物。在目标2中，将进行生化研究以检验Pol？合成DNA对偶（6-4）TT光产物，8-oxoG, edA， PdG和N2-dG BPDE。通过稳态动力学分析，我们将确定Pol？用于插入DNA损伤对面的核苷酸并进行随后的延伸反应，并且将进行生化研究以验证Pol？通过蛋白质模板导向机制将嘌呤插入到相反的DNA损伤中。我们在此提出的遗传和生化研究，对于描述TLS pol在促进复制过程中无错误和诱变病变旁路中的作用，以及对环境和细胞DNA损伤剂诱导的人类细胞诱变和致癌的遗传基础提供全面的了解，具有重要意义。我们对pol的主要无错误TLS模式的建议？,吗?， k, Rev1，和？可以预测这些Pol在癌症抑制中的作用，而Pol的TLS诱变模式？可以预测这种Pol在增强基因组不稳定性和致癌方面的作用。

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.