Analysis of the mechanism of ATP stimulation of calf thymus DNA .alpha.-polymerase

Abstract

Biochemical kinetic analyses of the ATP stimulation of the A2 form of calf DNA .alpha.-polymerase show that when DNA or primer termini are the variable substrates, maximum reaction velocity is independent of ATP concentration. When [deoxyribonucleoside triphosphate] concentration is the variable substrate, the apparent Km is invariant with ATP. The increase in the synthetic rate caused by ATP results from an improvement in synthesis initiation at primer termini. The effect of ATP on the DNA binding affinity of .alpha.-A2-polymerase was examined by using column chromatography. Passage of the polymerase through native DNA-cellulose at 70 mM ionic strength resulted in 40% binding of the enzyme. In the presence of 4 mM ATP, binding increased to 80%. In both cases, the bound polymerase could be eluted by a 370 mM ionic strength wash. An elution profile similar to that observed in the absence of ATP was obtained with 0.1 mM ATP, 4 mM GTP or 4 mM each of the nonhydrolyzable ATP analogs adenyl-5''-yl imidodiphosphate or adenosine 5''-O-(3-thiotriphosphate). The hydrolysis of the .gamma.-phosphate occurs at millimolar levels of ATP and leads to a higher affinity of polymerase for DNA. To distinguish the effects of ATP on RNA priming from those on DNA synthesis, products synthesized processively by .alpha.-A2-polymerase were sized by gel filtration. Essentially all products made on a gapped fd replicative form template in the presence of 4 dNTP and 4 mM ATP result from the extension of preexisting DNA primers. When rNTP other than or in addition to ATP are present, products primed de novo, approximately 30 nucleotides long, are observed. The length of processive synthesis of these products is unaffected by the presence of 4 mM ATP. At physiological concentrations (10 nM-10 .mu.M), the ATP analog P1,P4-di(adenosine-5'') tetraphosphate did not stimulate the synthetic rate of .alpha.-A2-polymerase. At millimolar concentrations moderate stimulation was observed.