The Bacillus subtilis model system will be utilized in the dissection of a DNA repair system responsible for handling certain types of environmental insults. Previous and preliminary results have demonstrated that the excision-repair system of B. subtilis is controlled by a limited (two) number of genes, is functional on a variety of bulky DNA lesions, and is important in the removal of lethal and pre-mutagenic lesions. This repair system will be characterized at the molecular level by the isolation and purification of the enzymes responsible for the removal of the DNA damage. Specifically, the two known genetic loci responsible for controlling this system will be cloned on a bivalent plasmid (capable of replicating and expressing in B. subtilis and in E. coli) vector. The proteins produced by these cloned genes will be sized and the genes will be manipulated to yield strains of B. subtilis and E. coli hyper-productive for these products. Using the proteins produced by the engineered bacteria, the enzymatic activity of these proteins (alone and complexed together) will be elucidated. The excision repair proteins will be purified and their mode of action (glycosidic vs. endonucletic) will be determined. This determination will be accomplished by a combination of in vitro complementation assays and experimentation involving ccc DNA and/or specifically damaged DNA. The purification of the protein complex responsible for excision-repair in B. subtilis will be accomplished by using a variety of processes which have been developed for other model systems. In addition, an extensive and complete mutagenesis procedure will be initiated in order to determine if all of the genes involved in this system have been identified. Special attention will be given to those genes which could have been previously missed because they code for an essential gene product. The completion of this project will provide valuable information on how organisms handle the lethal and pre-mutagenic damage done to their DNA by environmental hazards. It is important to note that the B. subtilis model is phenotypically similar to eukaryotes in its excision repair system. Accurate assessments of environmental risks can only be made after these repair processes are understood.
