Further Genetic and Enzymological Characterization of the Three Bacillus subtilis Deoxyribonucleic Acid Polymerases

Abstract

Bacillus subtilis has three DNA polymerases. Polymerase III is necessary for DNA replication while polymerase I is involved in DNA repair. Four independent mutations leading to loss of polymerase I activity were mapped by transformation between mdh and phoP. Since one of these mutant strains produces a heat-labile polymerase this locus is likely the polymerase I structural gene. Some polymerase III mutants fail to grow at elevated temperatures and have a greatly increased mutation rate. The structural genes for polymerases I and III are unlinked. Purified preparations of the three B. subtilis DNA polymerases have been characterized. Polymerases II and III catalyze a high rate of synthesis in the absence of a full deoxyribonucleoside triphosphate complement, the mechanism of which is probably a truncation of the normal synthetic process. Arabinosylcytosine triphosphate competitively inhibits polymerases II and III as well as DNA synthesis in toluene-treated cells. This analogue is incorporated by these polymerases. Single-stranded DNA is a potent inhibitor of polymerases II and III but not I. The B. subtilis polymerases are readily separable by column chromatography and respond differently to high ionic strength, heat, sulfhydryl reagents, arylazopyrimidines, and various templates. The differences and similarities of the three B. subtilis enzymes relative to the correspondingly numbered Escherichia coli enzymes are discussed.