Characterization of Bacillus subtilis ExoA protein: a multifunctional DNA-repair enzyme similar to the Escherichia coli exonuclease III

To discover the physiological role of the Bacillus subtilis ExoA protein, w hich is similar in amino acid sequence to Escherichia coli exonuclease III, an exoA::Cm disruption was constructed in the chromosomal DNA of B. subtil is. There was no clear difference in tolerance to hydrogen peroxide and alk ylating agents between the disruptant and the wild type strain. An expressi on plasmid of the ExoA in E. coli was constructed by inserting the exoA gen e into the expression vector pKP1500. The purified ExoA was used to clarify enzymatic characterizations using synthetic DNA oligomers as substrates. A DNA oligomer containing a 1', 2'-dideoxyribose residue as an AP site, a DN A-RNA chimera oligomer, and a 3' end P-32-labeled oligomer were synthesized . It has been shown that the ExoA has AP endonuclease, 3'-5' exonuclease, r ibonuclease H, and 3'-phosphomonoesterase activities. Thus, it has been con firmed that ExoA is a multifunctional DNA-repair enzyme in B. subtilis that is very similar to E. coli exonuclease III except that ExoA has lower 3'-5 ' exonuclease activity than that of E. coli exonuclease III.