Ordered sequential mechanism of substrate recognition and binding by KB cell DNA polymerase alpha.

We have used a steady-state kinetic approach in conjunction with direct velocity gradient sedimentation binding studies to examine the detailed steps that are involved in the recognition of DNA primer-template and dNTPs by near-homogeneous human DNA polymerase alpha. We demonstrate that the interaction of the polymerase with its substrates obeys a rigidly ordered sequential terreactant mechanism, with template as the first substrate, followed by primer as the second substrate and dNTP as the third. Although the binding of primer is prerequisite to the kinetically significant binding of dNTP, specification of which of the four dNTPs can then add to the enzyme is absolutely determined by base sequence of the template (the first substrate). The critical element in the proof of the ordered mechanism is the demonstration of the phenomenon of induced substrate inhibition; the presence of a dideoxy-terminated primer (dead-end inhibitor) induces substrate inhibition by dNTP which is absolutely restricted to the dNTP complementary to the template to which the blocked primer is annealed. This inhibition is kinetically com the demonstration of the phenomenon of induced substrate inhibition; the presence of a dideoxy-terminated primer (dead-end inhibitor) induces substrate inhibition by dNTP which is absolutely restricted to the dNTP complementary to the template to which the blocked primer is annealed. This inhibition is kinetically com the demonstration of the phenomenon of induced substrate inhibition; the presence of a dideoxy-terminated primer (dead-end inhibitor) induces substrate inhibition by dNTP which is absolutely restricted to the dNTP complementary to the template to which the blocked primer is annealed. This inhibition is kinetically competitive with 3' -hydroxyl-terminated (unblocked) primer and approaches 100% at saturating levels of the complementary dNTP. Direct binding studies document the specific and exclusive ability of complementary dNTPs to drive the polymerase into a stable dead-end complex with the proposed structure, enzyme.template.dideoxy primer.dNTP, thus corroborating the kinetic observations. Attempts to elucidate the order of product release from the enzyme by product inhibition studies have shown the polymerization reaction to be essentially irreversible and have thus been unsuccessful. On the basis the known processivity of KB cell DNA polymerase alpha, a preliminary model involving initial release of pyrophosphate is reasonable; however, the relationship between product release and the process of polymerase translocation remains obscure. All of the kinetic and sedimentation binding studies were performed on a variety of homopolymeric and natural heteropolymeric DNA substrates, and the consistency of the results establishes absolutely the qualitative identity of the general mechanism by which human DNA polymerase alpha recognizes and replicated polydeoxynucleotide primer-templates, regardless of their precise physicochemical nature.