Enzymatic determinants of DNA polymerase accuracy. Theory of coliphage T4 polymerase mechanisms

The specificity of base selection in DNA synthesis is affected by the DNA polymerase. This paper presents a detailed model of the enzymatic reaction steps involved in template-directed DNA synthesis by DNA polymerase possessing 3′ to 5′ exonuclease activity (the T4 coliphage enzyme and polymerases I, II and III of Escherichia coli are known examples). The central assumptions of the model imply that the strength of the hydrogen bonds formed between candidate bases and the template base provide the principal discrimination criteria for both the insertion and the “editing” excision of bases. We have performed a detailed analysis of this model, and of the stochastic, sequential, single-step process of insertion and excision, which it implies, and have compared the results with experimental data reported by Bessman et al. (1974a,b) for the DNA polymerase of T4. In the present version of the model, the presence of the editing exonuclease accounts for the enzyme's contribution to the accuracy of polymerization and all binding and reaction sites on the enzyme are insensitive to differences between bases or base-pairs. The Bessman data, describing the competitive incorporation of adenine and 2-aminopurine by five allelic polymerases and spanning a spectrum of mutation rates, are well fit by this theory, and permit us to predict the effects of changes in reaction conditions and in basepairing free energy on the outcome of similar experiments.