THE BIOCHEMISTRY OF DNA ALKYL PHOSPHOTRIESTERS

DNA 烷基磷酸三酯的生物化学

• 批准号: 3176098
• 负责人: DAVID E JENSEN
• 金额: $ 6.48万
• 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 1990-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3176098
• 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.

定量上最大的DNA氧损伤是由一个家族产生的 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以及 1-甲基-2-硝基-1-亚硝基胍位于主链磷酸基团上。 产率比鸟嘌呤 06 位高 2 至 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 这些烷基磷酸三酯对 DNA 的生物化学影响有多大？ 我正在考虑它们加剧约束力的可能性 蛋白质的作用是确保遗传物质的正确传递 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 长度和序列包含任何百分比的非对映体纯 乙基磷酸三酯。 鉴于磷酸三酯可能会干扰 定义蛋白质识别位点的 DNA 构象信号，I 将考虑这些损伤对 DNA 螺旋构象的影响 使用螺旋-螺旋转变实验的构象稳定性和 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。 内在结合常数和协同性的扰动 将利用色氨酸荧光来评估参数 淬火。 大肠杆菌聚合酶 I 的效率 将测试含磷酸三酯的模板。 我将检查 聚合率、三磷酸核苷周转率和核苷酸 利用标准方案和 HPLC 分析实现掺入保真度。 最后我将研究磷酸三酯在已知条件下的影响 lac UV5 启动子的接触位置对大肠杆菌 RNA 效率的影响 使用失败的起始测定来起始聚合酶信息。 这 启动子片段可以从头合成或通过以下方式产生 利用一些为寡核苷酸指导开发的方法 位点特异性诱变。