51. Exonucleolytic proofreading increases the accuracy of DNA synthesis by human lymphocyte DNA polymerase alpha‐DNA primase.
- Author
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Bialek, G., Nasheuer, H. P., Goetz, H., and Grosse, F.
- Abstract
DNA polymerase‐primase complex, isolated with an apparently undegraded alpha‐subunit, was immunoaffinity‐purified to near homogeneity from the human lymphoblast line HSC93. The undegraded state of the alpha‐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 phi X174am16 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 phi X174am16 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:Ttemplate mispairs, between 10(‐7) and 10(‐8) for dCMP:Ttemplate (1/35,000,000), dCMP:Atemplate (1/18,200,000) and dAMP:Gtemplate mispairs (1/16,500,000), and below 10(‐8) (1/100,000,000) for dTMP:Ttemplate, dGMP:Atemplate and dGMP:Gtemplate 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.(ABSTRACT TRUNCATED AT 250 WORDS) more...
- Published
- 1989
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