Exonucleolytic proofreading enhances the fidelity of DNA synthesis by chick embryo DNA polymerase-gamma.
The high fidelity of chick embryo DNA polymerase-gamma (pol-gamma) observed during in vitro DNA synthesis (Kunkel, T. A. (1985) J. Biol. Chem. 260, 12866-12874) has led us to examine this DNA polymerase for the presence of an exonuclease activity capable of proofreading errors. Highly purified chick embryo pol-gamma preparations do contain exonuclease activity capable of digesting radiolabeled DNA in a 3'----5' direction, releasing deoxynucleoside 5'-monophosphates. The polymerase and exonuclease activities cosediment during centrifugation in a glycerol gradient containing 0.5 M KCl. In the absence of dNTP substrates, this exonuclease excises both matched and mismatched primer termini, with a preference for mismatched bases. Excision is inhibited by the addition of nucleoside 5'-monophosphates to the digestion reaction. In the presence of dNTP substrates to permit competition between excision and polymerization from the mismatched primer, the exonuclease excises mismatched bases from preformed terminal mispairs with greater than 98% efficiency. The preference for excision over polymerization can be diminished by addition of either high concentrations of dNTP substrates or nucleoside 5'-monophosphates to the exonuclease/polymerase reaction. To determine if this exonuclease is capable of proofreading misinsertions produced during a normal polymerization reaction, a sensitive base substitution fidelity assay was developed based on reversion of an M13mp2 lacZ alpha nonsense codon. In this assay using reaction conditions that permit highly active exonucleolytic proofreading, pol-gamma exhibits a fidelity of less than one error for every 260,000 bases polymerized. As for terminal mismatch excision, fidelity is reduced by the addition to the synthesis reaction of high concentrations of dNTP substrates or nucleoside 5'-monophosphates, both hallmarks of exonucleolytic proofreading by prokaryotic enzymes. Taken together, these observations suggest that the 3'----5' exonuclease present in highly purified chick embryo pol-gamma preparations proofreads base substitution errors during DNA synthesis. It remains to be determined if the polymerase and exonuclease activities reside in the same or different polypeptides.
Kunkel
T A
TA
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
Soni
A
A
D000818:Animals; D002642:Chick Embryo; D003062:Codon; D004258:DNA Polymerase III / Q000378:metabolism*; D004260:DNA Repair; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000378:metabolism; D005092:Exonucleases / Q000378:metabolism*
D003062:Codon; D003854:Deoxyribonucleotides; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases