Purification and properties of the deoxyribonucleic acid polymerase induced by vaccinia virus.
The vaccinia virus-induced DNA polymerase has been purified about 500-fold from a cytoplasmic extract of vaccinia-infected HeLa cells. Analysis of the purified fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals a single polypeptide of 110,000 daltons, which is greater than 95% pure. This polypeptide co-sediments with polymerase activity through a glycerol gradient. The sedimentation coefficient of the enzyme is 6.3 S, and its Stokes radius is 4.6 nm. The molecular weight of the native enzyme derived from these values is 115,000. Vaccinia polymerase is therefore a single large polypeptide of 110,000 to 115,000 daltons. The purified fraction has no significant endonuclease activity, but a strong exonuclease activity co-purifies with polymerase activity through every step in the isolation. The polymerase and exonuclease activities are inactivated at 45 degrees C at the same rate. It is likely, therefore, that both activities are catalyzed by the same polypeptide. The exonuclease hydrolyzes DNA predominantly in the 3' leads to 5' direction, to produce 5' mononucleotides. The exonuclease degrades single-stranded DNA more rapidly than duplex DNA, and the rate of digestion of both single-stranded and double-stranded DNA increases as the size of the substrate decreases. Single-stranded circular DNA is a potent inhibitor of the exonuclease activity, but duplex circular DNA has no significant effect on its activity.
D004270:DNA, Circular; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000378:metabolism; D005092:Exonucleases / Q000378:metabolism; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D011487:Protein Conformation; D013379:Substrate Specificity; D014616:Vaccinia virus / Q000201:enzymology*
D004270:DNA, Circular; D004259:DNA-Directed DNA Polymerase; D003851:Deoxyribonucleases; D005092:Exonucleases