The objectives of the research are to understand mechanisms of DNA repair and mutagenesis. The cellular process of repair is important in maintenance of genetic information in correct context and expression format. We now know that processes associated with repair of DNA actually promote mutations in some cases. Mutagenesis of DNA can lead to genetic disease and is a primary step in the pathway of carcinogenesis. We will investigate the regulation and function of the uvrC gene of E. coli. This gene acts in excision repair of bulky damage in DNA. Our approach will be to capitalize on a bank of recombinant DNA constructs containing the structural gene with combinations of four 5' promoters. We will analyze lexA regulation and do site-directed mutagenesis to determine regulation. The overproduced gene product will be analyzed functionally in an in vitro reaction to determine its role in excision. We will take advantage of a group of mutants of E. coli which can survive without a functional DNA polymerase III. The conditional lethality of the polC mutation is reversed by an extragenic mutation. These mutants offer a unique resource to study the role of DNA polymerase III in repair and mutagenesis. The mutants are more sensitive to hydrogen peroxide than normal cells and we will use extracts from these mutants to develop an in vitro repair assay for hydrogen peroxide.
