THE BIOCHEMISTRY OF DNA ALKYL PHOSPHOTRIESTERS

DNA 烷基磷酸三酯的生物化学

• 批准号: 3176097
• 负责人: DAVID E JENSEN
• 金额: $ 6.52万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-07-01 至 1990-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3176097
• 关键词: DNA repair; Escherichia coli; autoradiography; binding proteins; chemical carcinogen; chemical carcinogenesis; diethylnitrosamine; enzyme mechanism; gel electrophoresis; high performance liquid chromatography; phosphoric ester; radiotracer

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.

数量上最大的DNA氧损伤产生的一个家庭， 1-烷基-1-亚硝基化合物致癌物，其中包括二甲基和 二乙基亚硝胺，甲基和乙基亚硝基脲， 1-甲基-2-硝基-1-亚硝基胍，在主链上是磷酸基团。 产率比鸟嘌呤的06位高2至5倍。 DNA 氧烷基化与致癌潜力相关。 目前还不知道 这些烷基磷酸三酯在多大程度上影响DNA的生物化学; 我在考虑他们是否有可能加剧 蛋白质的功能，以确保适当的遗传传递， 信息. 使用自动DNA合成仪和β-氰乙基 亚磷酰胺化学我建议合成已知的DNA聚合物 长度和序列含有任何百分比的非对映异构体纯 乙基磷酸三酯 认为磷酸三酯会干扰 定义蛋白质识别位点的DNA构象信号， 将考虑这些损伤对DNA螺旋构象的影响， 使用螺旋-卷曲转变实验的构象稳定性， 圆二色性 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。 内禀结合常数和协同性的扰动 将利用色氨酸荧光评价参数 淬火 E.大肠杆菌聚合酶I 将测试含磷酸三酯的模板。 我会检查 聚合速率、核苷三磷酸周转率和核苷酸 利用标准方案和HPLC分析的掺入保真度。 最后，我将研究磷酸三酯的影响， lac UV 5启动子的接触位置对E.杆菌RNA 聚合酶信息起始使用失败的起始测定。 的 启动子片段将从头合成或通过 利用一些开发的方法， 定点诱变。