THE BIOCHEMISTRY OF DNA ALKYL PHOSPHOTRIESTERS

数量上最大的DNA氧损伤是由一系列 1-烷基-1-亚硝基化合物致癌物，包括二甲基和亚硝基 二乙基亚硝胺、甲基和乙基亚硝脲和 1-甲基-2-硝基-1-亚硝基是磷酸基团的主链。 产率比鸟嘌呤的06位高2~5倍。脱氧核糖核酸 氧烷基化与致癌潜力相关。目前尚不清楚 这些烷基磷酸三酯在多大程度上影响DNA的生化； 我正在考虑这样一种可能性，即它们加剧了 蛋白质的作用是确保基因的正确传递 信息。使用自动DNA合成器和β-氰乙基 亚磷酰胺化学我建议合成已知的DNA聚合物 包含任意百分比的非对映异构体纯度的长度和序列 乙基磷三酯。认为磷酸三酯可能会扰乱 定义蛋白质识别位点的DNA构象信号，即 将考虑这些损伤对DNA螺旋构象的影响 螺旋-螺旋转变实验和构象稳定性的研究 圆二向色性。我将评估磷酸三酯对 单链结合蛋白T4噬菌体基因32蛋白的活性。 内禀结合常数和协作性中的微扰 参数将利用色氨酸荧光进行评估 淬火。大肠杆菌聚合酶I对细菌生长的影响 将测试含有磷酸三酯的模板。我会研究一下 聚合速率、核苷三磷酸周转率和核苷酸 结合使用标准协议和高效液相分析的保真度。 最后，我将研究放置在已知位置的磷三酯的影响。 Lac UV5启动子的接触位置对大肠杆菌RNA效率的影响 使用流产启动试验的聚合酶消息启动。这个 启动子片段要么从头合成，要么由 利用为寡核苷酸导向开发的一些方法 定点突变。

Quantitatively the greatest DNA oxygen lesion produced by a family of 1-alkyl-1-nitroso compound carcinogens, which includes dimethyl and diethylnitrosamine, methyl and ethylnitrosourea and 1-methyl-2-nitro-1-nitrosoguanidine, is at the backbone phosphate groups. The yield is 2-to 5-fold higher than at the 06-position of guanine. DNA oxygen alkylation correlates with carcinogenic potential. It is not known to what degree these alkyl phosphotriesters effect the biochemistry of DNA; I am considering the possibility that they aggravate the binding of proteins which function to insure the proper transmission of genetic information. Using an automatic DNA synthesizer and the Beta-cyanoethyl phosphoramidite chemistry I propose to synthesize DNA polymers of known length and sequence containing any percentage of diastereomerically pure ethyl phosphotriesters. With the view that phosphotriesters could perturb the DNA conformational signals which define protein recognition sites, I will consider the effects of these lesions on DNA helical conformation and conformational stability using helix-coil transition experiments and circular dichroism. I will assess the effects of phosphotriesters on the activity of the single strand binding protein, T4 phage gene 32 protein. Perturbations in the intrinsic binding constant and in the cooperativity parameter will be evaluated by taking advantage of tryptophan fluorescence quenching. The efficiency of E. coli polymerase I on phosphotriester-containing templates will be tested. I will examine the polymerization rate, nucleoside triphosphate turnover and nucleotide incorporation fidelity utilizing standard protocols and HPLC analysis. Finally I will study the effects of phosphotriesters placed at known contact positions of the lac UV5 promoter on the efficiency of E. coli RNA polymerase message initiation using the abortive initiation assay. The promoter fragment will be either synthesized de novo or produced by utilizing some of the methodology developed for oligonucleotide-directed site-specific mutagenesis.