The fidelity of DNA synthesis by the catalytic subunit of yeast DNA polymerase α alone and with accessory proteins

Abstract

The fidelity of DNA synthesis catalyzed by the 180-kDa catalytic subunit (p180 of DNA polymerase α from Saccharomyces cerevisiae has been determined. Despite the presence of a 3′ → 5′ exonuclease activity (Brooke et al., 1991, J. Biol. Chem., 266, 3005–3015), its accuracy is similar to several exonuclease-deficient DNA polymerase and much lower than other DNA polymerases that have associated exonucleolytic proofreading activity. Average error rates are 1/9900 and 1/12 000, respectively, for single base-substitution and minus-one nucleotide frameshift error; the polymerase generates detections as well. Similar erros rates are observed with reactions containing the 180-kDa subunit plus an 86-kDa subunit (p86), or with these two polypeptides plus two additional subunits (p58 and p49) comprising the DNA primase activity required for DNA replication. Finally, addition of yeast replication factor-A (RF-A), a protein preparation that stimulates DNA synthesis and has single-stranded DNA-binding activity, yield a polymerization reaction with 7 polypeptides required for replication, yet fidelity remains low relative to error rates for semiconservative replication. The data suggest that neither exonuceolytic proofreading activity, the β subunit, the DNA primase subunit substantially to base substitution or frameshift error discrimination by the DNA polymerase α catalytic subunit.