Purification and properties of wild-type and exonuclease-deficient DNA polymerase II from Escherichia coli.
Wild-type DNA polymerase II (pol II) and an exonuclease-deficient pol II mutant (D155A/E157A) have been overexpressed and purified in high yield from Escherichia coli. Wild-type pol II exhibits a high proofreading 3'-exonuclease to polymerase ratio, similar in magnitude to that observed for bacteriophage T4 DNA polymerase. While copying a 250-nucleotide region of the lacZ alpha gene, the fidelity of wild-type pol II is high, with error rates for single-base substitution and frameshift errors being < or = 10(-6). In contrast, the pol II exonuclease-deficient mutant generated a variety of base substitution and single base frameshift errors, as well as deletions between both perfect and imperfect directly repeated sequences separated by a few to hundreds of nucleotides. Error rates for the pol II exonuclease-deficient mutant were from > or = 13- to > or = 240-fold higher than for wild-type pol II, depending on the type of error considered. These data suggest that from 90 to > 99% of base substitutions, frameshifts, and large deletions are efficiently proofread by the enzyme. The results of these experiments together with recent in vivo studies suggest an important role for pol II in the fidelity of DNA synthesis in cells.
Cai
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Department of Biological Science, Hedco Molecular Biology Laboratories, University of Southern California, Los Angeles 90089-1340, USA.
Yu
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McEntee
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Kunkel
T A
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Goodman
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MF
D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D004257:DNA Polymerase II / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D005092:Exonucleases / Q000378:metabolism*; D016368:Frameshift Mutation; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids; D017354:Point Mutation; D017384:Sequence Deletion; D013698:Templates, Genetic; D010948:Viral Plaque Assay; D014764:Viral Proteins / Q000378:metabolism; D001616:beta-Galactosidase / Q000096:biosynthesis
D017931:DNA Primers; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; D004257:DNA Polymerase II; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases; D001616:beta-Galactosidase