KB CELL DNA POLYMERASE-α: MECHANISM OF PRIMER-TEMPLATE RECOGNITION BY THE CORE CATALYTIC UNIT

Abstract

ABSTRACT The catalytic properties of a homogeneous preparation of human KB cell DNA polymerase-a have been studied in detail. With respect to the enzyme's interaction with DNA, we have identified two kinetically distinguishable classes of sites on the enzyme for the binding of nucleic acid substrates. One of these is specific for single-stranded DNA (template site) while the second recognizes 3'-hydroxyl termini. Template binding is not dependent on the participation of 3'-hydroxyl termini. In contrast, the primer site appears to be able to bind a 3'-hydroxyl terminus only when the template site is occupied, and such binding is restricted to 3'-terminal residues that can base-pair with the template. Our data indicate that only minimal terminal complementarity (3 to 5 base pairs) is required. Spermidine is a specific effector of the primer-binding event and acts to destabilize the polymer-ase-DNA complex in such a way as to increase dramatically both the apparent Km for primer-template and the maximal velocity (V max) of the polymerization reaction. Kinetic data suggest that there are two template binding sites on each molecule of DNA polymerase-Q! and that these sites interact cooperatively (positively) by a simple allosteric mechanism. Evidence has also been obtained for two strongly cooperative primer-binding sites per molecule of enzyme.