title: Structural and enzymological characterization of the homogeneous deoxyribonucleic acid polymerase from Mycoplasma orale.
abstract: We have purified the DNA polymerase from Mycoplasma orale to homogeneity. The protein structure of the enzyme was declined by sodium dodecyl sulfate gel electrophoresis, which revealed a single band of 116 000 daltons that was coincident with the polymerase activity profile in the final step of DNA--cellulose chromatography, and by two-dimensional gel analysis, which demonstrated a single protein species at pI = 6.8 that was congruent with enzyme activity and contained the same 116 000 polypeptide. although severe enzyme aggregation occurs during nondenaturing gel electrophoresis, a monomer species can be resolved with a Mr of 140 000 by the Ferguson plot analysis. Gel filtration and velocity gradient centrifugation yield a Stokes radius of 4.8 nm and a sedimentation coefficient of 5.6 S, respectively, from which Mr values of 106 000--128 000 can be computed. The different size values suggest that the polymerase molecule is asymmetric. The purified enzyme has a specific activity of approximately 6 x 10(5) units/mg of protein and in completely devoid of exodeoxyribonuclease and endodeoxyribonuclease activities, at exclusion limits of 10(-4)--10(-6%) of the polymerase activity. The mechanism of polymerization is moderately processive, with an average of 14 +/- 4 nucleotides incorporated per binding event, and the "effective template length" on activated DNA is approximately 40 nucleotides.
mesh_terms: D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification / Q000378:metabolism*; D005092:Exonucleases / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D009174:Mycoplasma / Q000201:enzymology*; D013095:Spermidine / Q000494:pharmacology; D013096:Spermine / Q000494:pharmacology
chemical_list: D013096:Spermine; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases; D013095:Spermidine