VACCINIA VIRUS-DNA POLYMERASE - IN-VITRO ANALYSIS OF PARAMETERS AFFECTING PROCESSIVITY

The polymerization and proofreading activities of the vaccinia virus D NA polymerase reside within a 118-kDa catalytic polypeptide. We report here an investigation of the intrinsic processivity of this enzyme on both natural and homopolymeric DNA templates. Inclusion of the Escher ichia coli helix destabilizing protein allowed the viral enzyme, which lacks strand displacement activity, to utilize a singly primed M13 DN A template. In the presence of either 10 mM MgCl2 or 1 mM MgCl2 + 40 m M NaCl, synthesis was achieved in a highly distributive manner, RFII f ormation required a significant excess of enzyme, and less than or equ al to 10 nucleotides (nt) were added per primer-template binding event . The apparent rate of primer elongation varied with the enzyme/templa te ratio and reached a maximum of 8 nt/s. A similar lack of processivi ty was observed on a poly(dA(390))-oligo(dT(12-18)) template. In contr ast, highly processive synthesis was achieved on both templates in the presence of 1 mar MgCl2 and the absence of NaCl. A primer extension r ate of 30 nt/s was observed, and greater than or equal to 2000 nt were added per binding event. These studies suggest that the catalytic pol ypeptide of the vaccinia virus DNA polymerase will require accessory p rotein(s) to form a stable enzyme-template interaction and direct proc essive DNA synthesis under isotonic conditions in vivo.