REPLICATION OF DAMAGED DNA IN MAMMALIAN CELL EXTRACTS

DNA损伤的大多数遗传效应是在DNA过程中发生的

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.