Exonucleolytic proofreading by a mammalian DNA polymerase.
Porcine liver DNA polymerase gamma contains exonuclease activity capable of digesting DNA in the 3'----5' direction, releasing deoxyribonucleoside 5'-monophosphates. The exonuclease activity excises 3'-terminal bases from both matched and mismatched primer termini, with a preference for mismatched bases. Under polymerization conditions, mismatch excision by the exonuclease occurs prior to polymerization by polymerase gamma, and this excision can be inhibited by adding to the reaction a high concentration of dNTP substrates and/or nucleoside 5'-monophosphates. In an M13mp2-based reversion assay for detecting single-base substitution errors, porcine liver polymerase gamma is highly accurate; the estimated base substitution error rate is less than one error for each 500,000 bases polymerized. Lower fidelity is observed using reaction conditions that inhibit the exonuclease activity, strongly suggesting that the exonuclease proofreads errors during polymerization. However, in a forward mutation assay capable of detecting all 12 mispairs at a variety of template positions, certain base substitution errors are readily detected even using unperturbed polymerization conditions. Thus, for some errors, polymerase gamma is not highly accurate, suggesting that proofreading is not equally active against all mispairs. To examine if the polymerase and exonuclease activities are physically as well as functionally associated, both activities were monitored during purification by four procedures, each based on a different separation principle. The two activities copurify during chromatography using phosphocellulose, heparin-agarose, or double-strand DNA-cellulose, and during velocity sedimentation in a glycerol gradient containing 0.5 M KCl. These results suggest that the polymerase and exonuclease activities are physically associated. It remains to be determined if they reside in the same subunit.
Kunkel
T A
TA
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
Mosbaugh
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DW
D000818:Animals; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004258:DNA Polymerase III / Q000302:isolation & purification / Q000378:metabolism*; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008930:Mitochondria, Liver / Q000201:enzymology*; D009154:Mutation; D013552:Swine
D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases