Detection of the catalytic activities of DNA polymerases and their associated exonucleases following SDS-polyacrylamide gel electrophoresis.
A method is described to detect DNA polymerases and nucleases in homogeneous or crude enzyme preparations after electrophoresis in SDS-polyacrylamide gels(2) containing the appropriate template or substrate. DNA polymerases are electrophoresed in a gel containing gapped calf thymus DNA and after a renaturation treatment, the gel is incubated in a reaction mixture in which one deoxyribonucleoside triphosphate is [alpha-32P]-labelled. Incorporation of radioactivity into DNA is detected at the vicinity of the polymerase band by autoradiography. An associated nuclease activity can be measured after electrophoresis in a gel containing 32P-labelled gapped DNA, when nucleolytic digestion is seen as a clear band in the resulting autoradiogram. The gels can subsequently be stained with Coomassie blue to establish identical molecular weights of polymerase, nuclease and protein bands. Applications of this technique are discussed.
D001412:Bacillus subtilis / Q000201:enzymology; D004256:DNA Polymerase I / Q000378:metabolism; D004257:DNA Polymerase II / Q000378:metabolism; D004258:DNA Polymerase III / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology; D005092:Exonucleases / Q000378:metabolism*; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D008970:Molecular Weight; D012967:Sodium Dodecyl Sulfate
D012967:Sodium Dodecyl Sulfate; D004256:DNA Polymerase I; D004257:DNA Polymerase II; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases