IN VITRO ANALYSIS OF A DNA REPLICATION APPARATUS

DNA，作为细胞的遗传库，必须复制并 保持非常高的精确度。7种纯净品的组合 噬菌体T4的复制蛋白能够在体外复制DNA 准确度与体内DNA复制过程中观察到的准确度相当。我们 计划通过以下方式分析控制错误率所涉及的机制 使用该系统的相邻DNA序列。特别是，我们将 确定(A)相邻序列中的碱基对的数量 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的热稳定性之间的任何关系 序列和观察到的扰动程度以及(C)哪些蛋白质 有助于认识到由于错配而产生的扰动。 我们环境中的各种代理人不断地改变结构和 我们细胞中DNA的信息含量。有一个很大的电池 修复监测这种变化的机制，并纠正它们。 然而，其中一些改变逃脱了修复并导致了突变 和癌症。到目前为止，试图了解诱变的机制 由于致癌物利用了相对较低的准确性的酶作为 探测器。我们计划使用噬菌体T4的高度精确的复制复合体 作为一个探测器。致癌物产生的修饰脱氧核苷酸 与DNA的正常dNTP底物的相互作用将被合成并 纯净的。将检查这些改变的dNTP的配对属性 使用敏感测序和传染性分析。DNA模板 在天然DNA中的特定位置包含这些修饰的核苷酸将 是被建造的。这些修饰核苷酸的配对性质为 DNA复制过程中的模板残留物将在体外和 在活体内。

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.