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, which is similar in amino acid sequence to Escherichia coli exonuclease III, an exoA::Cm disruption was constructed in the chromosomal DNA of B. subtilis. There was no clear difference in tolerance to hydrogen peroxide and alkylating agents between the disruptant and the wild type strain. An expression plasmid of the ExoA in E. coli was constructed by inserting the exoA gene 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 DNA-RNA chimera oligomer, and a 3' end 32P-labeled oligomer were synthesized. It has been shown that the ExoA has AP endonuclease, 3'-5' exonuclease, ribonuclease H, and 3'-phosphomonoesterase activities. Thus, it has been confirmed 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.
Shida
T
T
Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Nagano, Japan. shida@giptc.shinshu-u.ac.jp
Ogawa
T
T
Ogasawara
N
N
Sekiguchi
J
J
D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D019757:Carbon-Oxygen Lyases / Q000235:genetics / Q000378:metabolism; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016914:Ribonuclease H / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D043211:Exodeoxyribonuclease V; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D016914:Ribonuclease H; D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D011088:DNA Ligases