IN VITRO ANALYSIS OF A DNA REPLICATION APPARATUS

DNA 复制装置的体外分析

• 批准号: 3272265
• 负责人: NAVIN K SINHA
• 金额: $ 14.84万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-08-01 至 1989-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3272265
• 关键词: DNA directed DNA polymerase; bacteriophage T4; carcinogens; frameshift mutation; gene expression; mutant; nucleic acid sequence; oligonucleotides; radiotracer; tissue /cell culture; virus DNA; virus genetics

DNA, being the genetic repository of the cell, must be duplicated and maintained with very high accuracy. A combination of 7 purified replication proteins of phage T4 is able to copy DNA in vitro with an accuracy comparable to that observed during DNA replication in vivo. We plan to analyze the mechanisms involved in control of error rates by neighboring DNA sequence using this system. In particular, we shall determine (a) the number of base pairs in the neighboring sequence that are perturbed due to T:G, T:C, T:T, A:A, A:C, A:G, G:A and G:G mispairs, (b)\any relationships between the thermal stability of the neighboring DNA sequence and the degree of observed perturbation and (c) which proteins contribute to the recognition of the perturbation due to a mispair. A variety of agents in our environment constantly change the structure and informational content of DNA in our cells. There is a large battery of repair mechanisms that monitor such alterations and correct them. Nevertheless, some of these alterations escape repair and lead to mutations and cancer. So far, attempts to understand the mechanisms of mutagenesis due to carcinogens have utilized enzymes of relatively low accuracy as probes. We plan to use the highly accurate replication complex of phage T4 as a probe. Modified deoxynucleotides resulting from carcinogen interaction with the normal dNTP substrates of DNA will be synthesized and purified. The pairing properties of these altered dNTPs will be examined using sensitive sequencing and infectivity assays. DNA templates containing these modified nucleotides in specific sites in natural DNA will be constructed. The pairing properties of these modified nucleotides as template residues during DNA replication will be examined both in vitro and in vivo.

DNA作为细胞的遗传储存库，必须被复制， 保持非常高的精度。 7个纯化的组合 噬菌体T4的复制蛋白能够在体外复制DNA， 与在体内DNA复制期间观察到的精确度相当。 我们 计划通过以下方式分析控制错误率的机制： 相邻的DNA序列使用该系统。 特别是，我们 确定（a）相邻序列中的碱基对的数目， 由于T：G、T：C、T：T、A：A、A：C、A：G、G：A和G：G错配而受到干扰， (b)\相邻DNA的热稳定性之间的关系 序列和观察到的扰动程度，以及（c）哪些蛋白质 有助于识别由于错对引起的扰动。 我们环境中的各种代理不断改变结构， 我们细胞中DNA的信息含量。 有一个大电池的 修复机制可以监控此类改变并进行纠正。 然而，这些改变中的一些逃避修复并导致突变 和癌症 到目前为止，试图了解诱变的机制， 由于致癌物的存在， probes. 我们计划使用噬菌体T4的高度精确的复制复合物 为探针 致癌物修饰的脱氧核苷酸 将合成与DNA的正常dNTP底物的相互作用， 提纯 这些改变的dNTPs的配对特性将被检查 使用灵敏的测序和传染性分析。 DNA模板 在天然DNA的特定位点含有这些修饰的核苷酸将 被建造。 这些修饰的核苷酸的配对性质如 DNA复制过程中的模板残基将在体外和 in vivo.