REPAIR OF DNA DAMAGED BY MODEL ENVIRONMENTAL CHEMICALS

该研究旨在确定遗传毒性和 化学致癌物诱导DNA损伤的突变效应 并研究它们修复的化学过程。 使用病毒DNA 转染系统和体外切割试验，我们已经发现， 该E.大肠杆菌，uvrA，B和C基因产物启动修复 由紫外线（UV）引起的各种各样的损伤， 化学致癌物，如N-乙酰氧基-2-乙酰氨基芴 N-羟基-2-乙酰氨基芴（N-OH-AF）， 苯并（a）芘二醇-环氧化物（BPDE）和DNA小沟 结合剂苏查如氨霉素和CC-1065。 这些病变 对DNA结构有不同的影响;纯化的UvrA，B， 而C蛋白则以不同程度的效率修复它们。 此外，解旋酶II对UVrABC活性有不同的影响 修复这些损伤。 我们会确定 这些影响的基础。 使用M13 mp 10 Lac+产生LAC -正向突变系统 我们检测到了一系列的突变， AAF-和AF-DNA aducts诱导的重组。 DNA 这些突变体中的序列变化表明：1） 其中两种加合物诱导相同碱基变化的序列， 2）SOS修复可能极大地改变突变谱 由DNA加合物诱导。 我们将确定 加合物形成和DNA序列对DNA复制的影响， 修复易感性和诱变，并将我们的发现应用于 研究三种基因突变的分子细节， 我们发现BPDE异构体具有不同的 遗传毒性和致突变性。 使用纯化的UvrA、B和C蛋白以及解旋酶II， 结合基因探针和Southern印迹技术， 开发了一种检测化学诱导DNA的技术 中国仓鼠卵巢aprt和dhfr基因的损伤 (CHO)细胞，并发现在aprt基因AF加合物发生 优先在非编码序列中。 我们建议用这个 测量差动感应和/或修复的方法 致癌物处理的哺乳动物细胞（野生型和 超敏突变体），研究基因活性的作用 （转录，扩增和甲基化）在DNA损伤和 修复，并评估差异修复在细胞毒性中的作用 和突变。

The proposed research aims to determine the genotoxic and mutational effects of chemical carcinogen-induced DNA damage and to study the chemistry of their repair. Using a viral-DNA transfection system and an in vitro incision assay we have found that the E. coli, uvrA, B and C gene products initiate the repair of a wide variety of lesions induced by ultraviolet light (UV), chemical carcinogens, such as N-acetoxy-2-acetylaminofluorene (NAAAF), N-hyroxy-2-acetylaminofluorene (N-OH-AF), benzo(a)pyrene diol-epoxide (BPDE), and DNA minor groove binding agents sucha as anthramycin and cc-1065. These lesions have different effects on DNA structure; the purified UvrA, B, and C proteins repair them with varying degrees of efficiency. Furthermore, helicase II has different effects on UvrABC activity in repairing these damages. We will determine the biochemcial basis of these effects. Using the M13mp10 Lac+ yields LAC - forward mutation system we have detected a broad spectrum of mutations and recombinations induced by AAF- and AF-DNA aducts. DNA sequence changes in these mutants show that 1) there are sequences in which both adducts induce identical base changes, and 2) SOS repair may greatly alter the mutation spectrum induced by DNA adducts. We shall determine the effects that adduct formation and DNA sequence have on DNA replication, repair susceptibility and mutagenesis and apply our findings to studying the molecular details of mutations induced by three BPDE isomers which we have found to have different genotoxicities and mutagenicities. Using purified UvrA, B and C proteins and helicase II, in combination with gene probes and Southern blot techniques, we have developed a technique to detect chemically induced DNA damage in the aprt and dhfr genes in Chinese hamster ovary (CHO) cells and found that in the aprt gene AF adducts occur preferentially in noncoding sequences. We propose to use this method to measure differential induction and/or repair in carcinogen treated mammalian cells (both wild type and hypersensitive mutants), to study the role of gene activity (transcription, amplification and methylation) in DNA damage and repair, and to assess the role of differential repair in cytotoxicity and mutation.