Although the excision repair mechanism for DNA damaged with UV radiation and carcinogenic chemicals was first described in E. coli many years ago and was shown to be dependent on the uvrA, uvrB and uvrC genes, the isolation and characterization the active enzyme has been remarkably difficult. These three genes have now been cloned, and the corresponding gene products have been identified. The overall goals of the proposed studies are first to purify the UVRA, UVRB and UVRC proteins to homogeneity, and then to characterize the individual proteins and the endonuclease activity of the holoenzyme that is expected to form when the three separate proteins are mixed together with damaged DNA. The specific approach will be to subclone the uvr genes in order to maximize the production of the three proteins. The UVRA, UVRB and UVRC proteins will then be purified by standard biochemical procedures and will be tested for activity in the complementation assay of Seeberg. Assuming that the purified proteins can be used to generate active UVRABC endonuclease, the specific sites of attack on DNA's damaged with UV or carcinogenic chemicals will be determined by using the methodology developed for DNA sequencing. When highly-purified active UVRABC endonuclease becomes available, we will attempt to reconstruct the entire nucleotide excision pathway with DNA polymerase and ligase to see whether the purified components can satisfactorily reproduce the intracellular response. While considerable progress has been made in the reconstruction of excision repair systems in which the initial event is the attack by a glycosylase followed by strand cleavage with an AP endonuclease, the major pathway of repair for pyrimidine dimers and other bulky lesions appears to involve the UVRABC system in E. coli, while similar multicomponent systems with a broad range of specificity are operative in eukaryotes such as yeast and humans. Thus, elucidating the UVRABC system may help to understand the even greater complexities of the eukaryotic nucleotide excision repair that removes most of the bulky lesions from DNA introduced by radiation and other carcinogens.
