Polymerization/excision kinetics of Escherichia coli DNA polymerase I. Stability in kinetic behaviour and variations of the rate constants with temperature and pH.

Abstract

1. The incorporation and excision kinetics of Escherichia coli DNA polymerase I in the replication of (dA)n with dUTP and (dI)n with dCTP was studied at various temperatures and pH. When the incorporation/excision ratio (dy/dx) is plotted against the concentration of deoxynucleoside triphosphate [S] two kinds of curves are obtained. With (dI)n, dy/dx increases with [S], then reaches an asymptotic value. This behaviour, consistent with a kinetic amplification or kinetic proofreading mechanism, is observed at all temperatures and pH. With (dA)n, dy/dx increases with [S] but in a convex, instead of a concave manner. In this case, we approximated the curves by straight lines at the origin, in conformity with the prediction of the frayed-unfrayed model. Again a single behaviour is consistently observed at all temperatures and pH. 2. The data were analyzed in terms of ratios or products of three kinetic constants: ki for incorporation, ke for excision and, in the (dI)n system, theta for the processing of the incoming dNTP. The dNMP production in the (dI)n system is dominated by the ke theta term which increases with temperature and pH. Temperature influences excision more than incorporation, the net result being a linear decrease of ki/ke with temperature. The effect is more pronounced with (dI)n than with (dA)n and is probably related to the stability of the template-primer complex. The ki/ke term shows a bell-shaped dependency with pH in the (dI)n system. With (dA)n it remains constant between pH 7.5 and 8.5 then decreases with a transition midpoint at pH 9.0. We suggest that the pH profiles may reflect the ionization of the template in the first case, and of the substrate in the second.