REPLICATION OF DAMAGED DNA IN MAMMALIAN CELL EXTRACTS

DNA损伤的大部分遗传效应在DNA过程中受到影响 复制。DNA中的损伤阻止复制：然而，随后， 修复机制使积木得以绕过。其中一个主要的 哺乳动物细胞中复制后修复途径通过从头开始发生 综合。这一途径可能与诱导修复过程有关。 并观察到几种病毒的突变。我们建议研究 小鼠和中国人提取物的体外复制 紫外线和N-乙酰氧基-AAF对金黄地鼠细胞DNA的损伤 DNA序列水平上的产物。我们将在以下方面比较合成 未经处理的细胞的提取物与紫外线预处理的细胞的提取物 辐射以检查合成受损的任何可诱导的变化 DNA使用0X174和细小病毒DNA作为模板，我们将定位 准确的区块位置，量化终止和调查能力 进行跨病变合成。我们将检查以下参数 导致病变终止或绕过病变，尤其是与 用紫外线对细胞进行前处理。任何跨病变合成都将是 检查其效率和核苷酸插入的特异性 在损坏的地方。这一试验性战略将提供一座桥梁 在以前的体内方法和体外实验中纯化的 聚合酶，以加深我们对所涉及的过程的理解 在复制诱变剂时破坏DNA。

Most of the genetic effects of DNA damage are effected during DNA replication. Lesions in the DNA block replication: subsequently, however, repair mechanisms allow the blocks to be circumvented. One of the major post-replication repair pathways in mammalian cells occurs by de novo synthesis. This pathway may be related to the inducible process of repair and mutagenesis observed with several viruses. We propose to examine replication as carried out in vitro by extracts from mouse and Chinese Hamster cells of DNA damaged by UV and N-acetoxy-AAF, analyzing the products at the level of the DNA sequence. We will compare synthesis in extracts from untreated cells with those from cells pre-treated with UV irradiation to examine any inducible changes in synthesization damaged DNA. Using 0X174 and paravovirus DNA as templates we will locate the precise sites of blocks, quantitate termination and investigate the ability to carry out trans-lesion synthesis. We will examine the parameters that lead to termination at or by-pass of lesions, particularly in relation to pre-treatment of cells with UV. Any trans-lesion synthesis will be examined as to its efficiency and the specificity of nucleotide insertion at the sites of damage. This experimental strategy will provide a bridge between previous in vivo approaches and in vitro experiments with purified polymerases in order to further our understanding of the processes involved in the replication of mutagen damaged DNA.