Deoxyribonucleic Acid Polymerase II of Escherichia coli: I. THE PURIFICATION AND CHARACTERIZATION OF THE ENZYME

Abstract

Abstract DNA polymerase II has been purified 27,000-fold in high yield by alumina grinding, polyethylene glycol-dextran phase partition, and chromatography on diethylaminoethyl-cellulose and phosphocellulose columns from Escherichia coli pol A- (DNA polymerase I negative strain) endo I-. This enzyme activity resembles DNA polymerase I described by Kornberg. (a) It requires as template DNA either treated with exonuclease III or partially degraded with DNase I; (b) the activity is completely dependent on the presence of Mg2+ and all four deoxynucleoside triphosphates; (c) like DNA polymerase I, DNA polymerase II is inhibited by pyrophosphate and catalyzes both pyrophosphorolysis of DNA and a DNA-dependent pyrophosphate exchange into deoxynucleoside triphosphates. Unlike DNA polymerase I, DNA polymerase II activity is inhibited by high salt concentration or reagents which react with sulfhydryl groups. DNA polymerase II is not inhibited by antibodies directed against DNA polymerase I and the two polymerases are separated by phosphocellulose chromatography or by sedimentation in glycerol gradients. DNA polymerase II has an exonuclease activity which preferentially degrades single stranded DNA in the 3' to 5' direction. In contrast, DNA polymerase I has two exonuclease activities, one acting 3' to 5' on denatured DNA, the other acting 5' to 3', on native DNA. DNA polymerase II has a sedimentation rate in high or low ionic strength and a migration on gel filtration consistent with a molecular weight of approximately 120,000. Based upon the extent of purification there are fewer than 17 molecules of DNA polymerase II per bacterium. This amount of enzyme can account for the incorporation into DNA of 800 deoxynucleotide residues per min per cell under optimal assay conditions. The in vivo rate of DNA synthesis is approximately 3 x 105 nucleotides incorporated into DNA per min per bacterium. DNA polymerase II is found in pol A+ cells at approximately the same concentration observed in pol A- cells. The enzyme has been isolated from septation mutants of E. coli which produce DNA-negative cells (minicells). DNA polymerase II is present in minicells at approximately half the specific activity observed in the whole cells. A similar distribution of DNA polymerase I has been reported previously for DNA negative cells. These results and those concerning the mode of action of DNA polymerase II presented in the accompanying paper (Wickner, R. B., Ginsberg, B., and Hurwitz, J. (1972) J. Biol. Chem., 247, 498) suggest that this enzyme is probably not involved in the replication of DNA but may have a repair function.