Mechanisms for induction and suppression of spontaneous and induced mutations

烷化剂是强效诱变剂和致癌物，有时会导致细胞死亡。烷基化试剂的这些作用主要归因于DNA中各种烷基化碱基的形成。其中，O^6-甲基鸟嘌呤似乎是诱发突变和癌症的主要原因。许多生物体都具有一种酶，即O^6-甲基鸟嘌呤-DNA甲基转移酶，它可以修复DNA中的O^6-甲基鸟嘌呤。它将甲基化DNA中的甲基转移到酶分子本身的半胱氨酸残基上。在大肠杆菌中，甲基转移酶（Ada蛋白）和其他参与DNA烷基化损伤修复的酶在细胞对相对低剂量的烷基化剂的反应中被诱导。这一过程被称为适应性反应，由ada基因控制，其产物既是一种修复酶，也是一种转录调节因子。研究表明，约五分之一的人类肿瘤细胞系表现为Mer^-表型，而Mer^-菌株缺乏甲基转移酶活性。通过cDNA转染，我们能够获得具有极高水平的O^6-甲基鸟嘌呤-DNA甲基转移酶活性的细胞系。用聚合酶链反应（PCR）技术从次生代谢物中扩增出甲基转移酶的cDNA，并测定了其核苷酸序列，为阐明乙酰氨基芴（AAF）化学诱变的分子基础，研究了AAF对DNA复制的影响。E.用一种新的方法分析了大肠杆菌DNA聚合酶Ⅲ在模板DNA上的一个特定位点的单一AAF加合物损伤。我们发现，DNA聚合酶III在模板加合位点前一个核苷酸处终止DNA合成，没有明显的旁路复制发生。此外，我们获得的证据表明，模板DNA的结构改变的AAF加合物衰减链的伸长。

Alkylating agents are potent mutagens and carcinogens and sometimes cause cell death. These effects of alkylating agents are mainly attributed to the formation of various alkylated bases in DNA. Among them, O^6-methylguanine appears to be most responsible for induction of mutations as well as of cancers. Many organisms possess an enzyme, O^6-methylguanine-DNA methyltransferase, that repairs O^6-methylguanine in DNA. It transfers a methylgroup from methylated DNA to a cysteine residue of the enzyme molecule itself. In Escherichia coli the methyltransferase enzyme (Ada protein) and other enzymes involved in repair of alkylation lesions in DNA are induced on response of cell to a relatively low dose of alkylating agent. This process, termed the adaptive response, is controlled by the ada gene, whose product is a repair enzyme as well as a transcriptional regulator.It has been demonstrated that about one-fifth of human tumor cell lines show the Mer^- phenotype, and that the Mer^- strains are deficient in the methyltransferase activity. By cDNA transfection, we were able to obtain a cell line possessing an extremely high level of O^6-methylguanine-DNA methyltransferase activity. The cDNA for methyltransferase was recovered from the secondary transformant by the polymerase chain reaction and its nucleotide sequence was determined.To elucidate molecular basis of the chemical mutagenesis by acetylaminofluorene(AAF), effects of this compound on the DNA replication were studied. Dynamics of E. coli DNA polymerase III on template DNA with a single AAF adduct lesion at a particular site was analized by a method newly developed in this work. We found that the DNA polymerase III stopped DNA synthesis at one nucleotide before the adduct site on the template and no obvious bypass replication occured. Further, we obtained evidence that a structural alteration of the template DNA by the AAF adduct attenuates the chain elongation.