Abstract
The contribution of base-pair stability to the fidelity by which the Klenow fragment of Escherichia coli DNA polymerase I copies the alternating DNA copolymers, polydeoxyinosine-deoxycytosine (poly{dI-dC}) and polydeoxyguanine-deoxycytosine (poly {dG-dC}) has been analyzed. Comparative studies of these DNA templates and substrate dNTPs (dITP, dGTP and dCTP) that base-pair by forming two (I=C) or three (G(TBOND)C) hydrogen bonds were possible. In addition, the effect of base-pair stability on DNA polymerase activity when the 3' to 5' exonuclease activity was inhibited by nucleoside monophosphates (5'NMP) was studied. The results of this study support a proposed model of the active center of DNA polymerase I in which the sites of excision and incorporation are independent (Que, B.G., et al. (1978) Biochemistry 17, 1603-1606). This study was further extended to include the influence of nearest neighbor base-pair stability on substitution errors during DNA synthesis in vitro. The results demonstrate that changing nearest neighbor base-pairs from I=C to G(TBOND)C can increase dTMP misincorporation greater than 30-fold. Specifically, the base-pair stability of the nucleotide incorporated following a misinsertion was found to influence the proofreading frequency of the enzyme. Similarly, an enhancement in misincorporation frequency as large as 30-fold was observed by selectively inhibiting the 3' to 5' exonuclease activity of the enzyme. Quantitation of nucleotide turnover and stable misincorporation revealed that the 3' to 5' exonuclease activity is responsible for a greater than 60-fold increase in the fidelity of nucleotide incorporation into poly{dI-dC} in some cases. As expected, when the following nucleotide forms a stable G=C base-pair in either poly{dI-dC} or poly{dG-dC}, the contribution of proofreading to fidelity is decreased.