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REPLICATION OF DAMAGED DNA IN MAMMALIAN CELL EXTRACTS

哺乳动物细胞提取物中受损 DNA 的复制

基本信息

批准号：
3174876
负责人：
PETER D MOORE
金额：
$ 13.51万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
1983
资助国家：
美国
起止时间：
1983-09-01 至 1988-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3174876
关键词：
DNA repair acetylaminofluorene autoradiography gel electrophoresis gene mutation radiotracer tissue /cell culture ultracentrifugation

项目摘要

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.

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照射以检查合成中的任何诱导变化是否受损 DNA. 使用0X 174和副病毒DNA作为模板，我们将定位精确的阻断位点，定量终止并调查能力进行跨病变合成。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。