Studies on the mechanism of DNA polymerase alpha. Nascent chain elongation, steady state kinetics, and the initiation phase of DNA synthesis.

A detailed study of the mechanism of nascent chain elongation and of steady state kinetics of purified mouse DNA polymerase alpha has been conducted. Polymerization was examined using a model replication system of poly(dT) as template, oligo(rA) as primer, and dATP as nucleotide substrate, and the probability of chain termination was determined by measurement of the precise chainlength of the products. Reactions were conducted under conditions where products were not utilized as primer. Product chainlength analysis indicated that alpha-polymerase acted in a processive fashion, elongating the primer by the stepwise addition of up to 20 dAMP residues before dissociating. The probability of termination after each dAMP addition depended upon the chainlength of the product and upon the presence of several agents; spermine, spermidine, putrescine, nalidixic acid, or PPi caused a marked increase in termination after the first dAMP addition, and conversely, mouse helix destabilizing protein-1 caused the enzyme to continue extending the same product chain until 18 to approximately 35 dAMP residues had been added. From these and other data, it is concluded that the kinetic mechanisms of termination after the first dAMP addition and after subsequent dAMP additions are different. With this information on how alpha-polymerase elongates a nascent primer(dA)n molecule, a kinetic model and appropriate steady state rate equations were obtained for analysis of substrate initial velocity data and termination probabilities. The substrate kinetic patterns and PPi product inhibition results were consistent with the ordered Ter Ter mechanism Bi Uni Uni Bi Ping Pong proposed in the model, and the model also permits a rational explanation for the differences in termination probability and for the fact that substrate initial velocity plots were linear even though multiple residues of dATP combined with the enzyme during each catalytic cycle. In addition, the results suggest that a rate-limiting step in the steady state occurs at the transition between initiation and elongation, and that higher levels of template.primer increase the rate of this step. This secondary effect of template.primer is discussed in relation to other polymer-forming enzymes, and various kinetic mechanisms which require the presence of two template.primer-binding sites, effector and catalytic, are discussed for their fit to the experimental data.