A Deoxyribonucleic Acid Polymerase from Micrococcus luteus (Micrococcus lysodeikticus) Isolated on Deoxyribonucleic Acid-Cellulose

A deoxyribonucleic acid polymerase from Micrococcus luteus has been isolated by allowing it to bind to its substrate, DNA, attached to a solid matrix of cellulose and subsequent detachment from the DNA by high salt concentrations. This approach has permitted the isolation of a highly purified enzyme by easy and reproducible methods from a partially purified preparation in which the polymerase is only a minor constituent. The enzyme preparations are quite active with DNA primers, almost entirely free of endo- or exonucleases, and extremely stable in the presence of DNA and other nucleotide polymers, or in high salt solution. At low salt concentrations, however, activity is rapidly lost. The active polymerase in high salt has a sedimentation rate of about 7 S. Kinetic studies indicate that the interactions between the polymerase-DNA structure and the deoxynucleoside triphosphates and Mg++ or Mn++ are complex. It can be calculated from DNA saturation data that there are only one or two sites of initiation on helical linear DNA molecules, and that reinitiation on a synthetic complex does not occur. DNA synthesis stops when a doubling of the added primer is reached.