Abstract
During catalysis by homogeneous procaryotic DNA polymerases, nucleoside monophosphates are generated by a 3' leads to 5'-exonucleolytic activity. Using Escherichia coli DNA polymerase I and poly[d(A-T)] as a template, the contribution of this activity to the fidelity of DNA synthesis has been evaluated by three different criteria. 1) The ratio between the rates of monophosphate generation and incorporation of the noncomplementary nucleotide with Mg2+ as an activating cation was 0.6 +/- 0.6, which is insufficient to account for the high fidelity of polymerization. 2) Inhibition of polymerization by pyrophosphate fails to diminish fidelity, although some kinetic models suggest that optimal error correction via monophosphate release requires the polymerization reaction to be strongly driven by pyrophosphate release. 3) The addition of deoxynucleoside monophosphates in concentrations as great as 10 mM to the reaction mixture does not alter the fidelity of DNA synthesis. These observations argue against the kinetic proofreading mode to account for the fidelity of E. coli DNA polymerase I when copying poly[d(A-T)] in a Mg2+-activated reaction. Furthermore, they suggest that the polymerase may enhance specificity at the base-selection step. However, the 3' leads to 5' exonuclease plays a larger role when the polymerase is activated with Mn2+ and may also be important in copying natural DNA where lower error rates are observed in vitro.
Highlights
During catalysis by homogeneous procaryotic DNA in free energy between correct and incorrect Watson-Crick polymerases, nucleoside monophosphates are gener- base pairings is a t most only[2] to 3 kcal/mol
E . coli polymerase I (Pol I) is highly accurate in copying poly[d(A-T)]; the frequency of [3H]dGTP misincorporation with Mg2+with different batches of Pol I and labeled substrates varied from 1/15,000 to 1/80,000.in replicate determinations with the same template,substrate, and enzyme preparation, the error rate varied by less than 5%.The rate of incorporation of the complementary and noncomplementary nucleotide is linear for at least 30 min of incubation using concentrations of Pol I between 0.1 and 10 nM
With [a-"PIdATP as thelabeled substrate, the ratoef polymerization in the fidelity assay approximates 60 mol of total nucleotides incorporated/mol of enzyme protein/min. This rate is less than that reported under conditions in which all components of the reaction are present in saturating amounts, 200-1000 nucleotides per min (15)
Summary
E . coli polymerase I (Pol I) is highly accurate in copying poly[d(A-T)]; the frequency of [3H]dGTP misincorporation with Mg2+with different batches of Pol I and labeled substrates varied from 1/15,000 to 1/80,000.in replicate determinations with the same template,substrate, and enzyme preparation, the error rate varied by less than 5%.The rate of incorporation of the complementary and noncomplementary nucleotide is linear for at least 30 min of incubation using concentrations of Pol I between 0.1 and 10 nM. Effect of nucleoside monophosphates on the 3‘ 4 5’ exonuclease presented evidence that this template is contaminated with activity of E. coli DNA polymeraseI only 1 in 2 x IO6 mol of dGMP/mol of total nucleotide and. 1/4,600 1/4,400 1/6,500 1/3,800 1/5,600 therate of noncomplementarymonophosphategeneration (see Tables I and 111).under the conditionsof Byrnes et al (43), kinetic competition is drastically altered to favor monophosphate generation, and the exonuclease may play a greater role in fidelity.Since their error rates aaret least 100fold higher than ours, we argue that the additional fidelity obtained under morpehysiologically relevant conditions is not explained by monophosphate generation. Would lead to greater insertion specificity due to increased discrimination energybetweencorrectandincorrect E T M intermediates
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