REPLICATION OF DAMAGED DNA IN MAMMALIAN CELL EXTRACTS

大多数DNA损伤的遗传效应是在DNA损伤过程中发生的。 复制的 DNA中的损伤阻止复制：然而，随后， 修复机制允许绕过障碍。 的一个主要 哺乳动物细胞中的复制后修复途径通过从头开始 合成. 该通路可能与诱导性修复过程有关 并在几种病毒中观察到诱变。 我们建议研究 用小鼠和中国人的提取物进行体外复制 用紫外线和N-乙酰氧基-AAF对细胞DNA进行损伤， DNA序列水平上的产物。 我们将比较合成在 未处理细胞的提取物与UV预处理细胞的提取物 辐射检查任何可诱导的变化，在合成受损 DNA. 使用0X 174和副病毒DNA作为模板，我们将定位 精确的组织块位点，定量终止并研究 进行跨病变合成。 我们将检查参数， 导致在病变处终止或绕过病变，特别是与 用UV预处理细胞。 任何跨病变合成将是 检查其效率和核苷酸插入的特异性 在损坏的地方。 这一实验性策略将为 在先前的体内方法和体外实验之间， 聚合酶，以进一步了解所涉及的过程 在复制诱变剂损伤的DNA中起作用。

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.