THE BIOCHEMISTRY OF DNA ALKYL PHOSPHOTRIESTERS

DNA 烷基磷酸三酯的生物化学

• 批准号: 3176096
• 负责人: DAVID E JENSEN
• 金额: $ 6.61万
• 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 1987-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3176096
• 关键词: DNA repair; Escherichia coli; autoradiography; chemical carcinogen; chemical carcinogenesis; gel electrophoresis; high performance liquid chromatography; phosphoric ester; radiotracer

The alkylation of phosphate group oxygens is quantitatively among the major DNA lesions produce in vitro and in vivo by the methylating and ethylating nitroso compounds. The role of these DNA phosphotriesters in the potent carcinogenic and mutagenic activity of these compounds in unknown. DNA phosphotriesters are chemically stable under physiological conditions and persist for relatively long periods in treated cells. There are indications from studies utilizing oligonucleotides that phosphotriesters may influence DNA conformation and conformational stability and also decrease the net rate of DNA polymerization by E. coli polymerase I. We propose experiments which will clearly demonstrate the influence of phosphotriesters on aspects od DNA biochemistry at the polymer level and potentially illustrates the part played by these lesions in the cellular response to alkylating nitroso compound damage. The synthetic DNA homopolymer poly(dT) can be methylated such that better than 98 percent of the alkyl groups are covalently bound to phosphate oxygens; we intend to utilize this modified singlestranded polymer and also the B-form helical duplex it forms with poly(dA) as the substrates in our experiments. We will determine the effects of phosphotriesters on DNA conformation and conformational stability by generating polymer mixing curves, by studying circular dichroism spectra and changes in these spectra with temperature and by observing thermally-induced helix-coli transitions done as a function of monovalent salt concentration. We propose also to study the influences of phosphotriesters in two well characterized DNA-protein interaction systems in which function is expected to be very sensitive to the integrity of the DNA sugar-phosphate backbone. We hope to verify that these lesions decrease the rate of polymerization by E. coli polymerase I and intend to test whether this pertubation is due perhaps to the stalling of polymerization or to changes in processivity. We will also determine whether DNA phosphotriesters induce nucleotide misincorporation. Finally we will consider the effect of phosphotriesters on the cooperative and contiguous binding characteristics of bacteriophage T 4-coded gene 32 protein, the prototype single-stranded DNA binding protein intimately involved in DNA replication, recombination and repair.

磷酸基团氧的烷基化在数量上是主要的反应之一。 DNA损伤在体外和体内产生的甲基化和乙基化 亚硝基化合物 这些DNA磷酸三酯在有效的 这些化合物的致癌和致突变活性未知。 DNA 磷酸三酯在生理条件下是化学稳定的， 在处理的细胞中持续相对长的时间。 有 使用磷酸三酯的寡核苷酸的研究表明， 可能影响DNA构象和构象稳定性， 降低E.大肠杆菌聚合酶I。 我们 提出实验，将清楚地证明的影响 磷酸三酯在聚合物水平上对DNA生物化学的影响， 这可能说明了这些病变在细胞中所起的作用， 对烷基化亚硝基化合物损伤的反应。 合成DNA 均聚物聚（dT）可以被甲基化，使得超过98%的聚（dT）可以被甲基化， 烷基与磷酸氧共价结合;我们打算 利用这种改性的单链聚合物和B型螺旋 在我们的实验中，它与作为底物的poly（dA）形成双链体。 我们 将确定磷酸三酯对DNA构象的影响， 通过生成聚合物混合曲线，通过研究 圆二色光谱和这些光谱随温度的变化 通过观察热诱导的螺旋-大肠杆菌转变， 单价盐浓度的函数。 我们亦建议研究 磷酸三酯在两种已充分表征的DNA-蛋白质中的影响 交互系统，其中的功能预计是非常敏感的， DNA糖磷酸骨架的完整性 我们希望证实， 这些损伤降低了E.大肠杆菌聚合酶I 并打算测试这种扰动是否可能是由于失速 聚合或持续合成能力的变化。 我们还将确定 DNA磷酸三酯是否诱导核苷酸错误掺入。 最后 我们将考虑磷酸三酯对合作社的影响， 噬菌体T4编码基因32的连续结合特性 蛋白质，原型单链DNA结合蛋白密切 参与DNA复制、重组和修复。