Exonucleolytic proofreading increases the accuracy of DNA synthesis by human lymphocyte DNA polymerase α-DNA primase

DNA polymerase-primase complex, isolated with an apparently undegraded α-subunit, was immunoaffinity-purified to near homogeneity from the human lymphoblast line HSC93. The undegraded state of the α-subunit was monitored by Western-blot analysis of crude cellular extracts and all active fractions obtained during purification. The human polymerase-primase consists of four subunits with molecular weights of 195, 68, 55 and 48 kd. The fidelity of the polymerase-primase in copying bacteriophage ΦX174am16 DNA in vitro was determined by measuring the frequency of production of different revertent phages. The overall accuracy was between 4 x 10^-6 and 10 x 10^-6. This value reflects the spontaneous mutation frequency of ΦX174am16 phages in Escherichia coli, and is 10- to 20-fold higher than the accuracy of a conventionally purified enzyme from calf thymus. The frequencies of base pairing mismatches, estimated from pool bias measurements, were 3.5 x 10^-7 (1/2 880,000) for dGMP:T(template) mispairs, between 10^-7 and 10^-8 for dCMP:T(template) (1/35,000,000), dCMP:A(template) (1/18,200,000) and dAMP:G(template) mispairs (1/16,500,000), and below 10^-8 (1/100,000,000) for dTMP:T(template), dGMP:A(template) and dGMP:G(template) mispairs. In contrast to previous preparations, the intact polymerase-primase possesses a 3'→5' exonuclease activity. This exonuclease removes both matched and mismatched 3'-OH ends, with a preference for mismatched bases. Fidelity was reduced 8-fold by increasing the concentration of the next nucleotide following the incorporated mismatch nucleotide. Upon replacing dGTP by its phosphorothioate analogue at equimolar concentrations of the four nucleoside triphosphates, a 2-fold increase in the number of revertants was observed; biasing dGTPαS 9- and 30-fold over dATP and dCTP, respectively, led to a 50-fold increase in the number of revertants. Taken together, these observations suggest that the 3'→5' exonuclease present in immunoaffinity purified human polymerase-primase proofreads nucleotide misinsertions during DNA synthesis. The exonuclease contributes at least one to two orders to magnitude to the high fidelity characteristics of the intact polymerase-primase complex.