The partial purification and characterization of DNA polymerase activity from free living Ṟẖi̲ẕo̲ḇi̲u̲m̲ j̲a̲p̲o̲ṉi̲c̲u̲m̲

DNA polymerase activity from free living Rhizobium japonicum was partially purified and characterized. The enzyme was isolated from late log phase cultures by extraction in a French pressure cell, phase separation and anion exchange chromatography on DEAE cellulose. Two peaks of DNA polymerase activity were partially separated by anion exchange chromatography. The molecular weight of the polymerase in the first peak was estimated to be approximately 1.0 x 10⁵ daltons by sedimentation in glycerol gradients. The two DNA polymerase activities were inhibited by l0% ethanol, stimulated by 4 mM N-ethylmaleimide and were incapable of utilizing the primed single stranded template, poly dA-01igo dT_{12-18. Each enzyme was observed to exhibit the highest activity in the absence of KCl in the reaction mixture and both enzymes exhibited a similar pattern of inhibition in response to the presence of increasing concentrations of KCl in the reaction mixture. Each of the two DNA polymerases lost approximately 90% of its activity when assayed after a 10 minute preincubation at 35°C. These results suggest that the two activities may be two forms of the same enzyme. When the polymerase in the second peak was rechromatographed on DEAE cellulose, an increased amount of activity was recovered in the position of the first peak, suggesting that there was a conversion of the second form}

of the enzyme to the first form.