PROKARYOTIC AND EUKARYOTIC DNA-POLYMERASES .1. THE ROLE OF INTERNUCLEOTIDE PHOSPHATES IN THE BINDING PROCESS OF OLIGONUCLEOTIDE PRIMER WITH THE ENZYME

  • 1 January 1987
    • journal article
    • research article
    • Vol. 13  (1) , 45-57
Abstract
The mechanism of binding and elongation of the oligothymidylate primers in the systems of the DNA polymerase .alpha. from human placenta and DNA polymerase I from Escherichia coli with the poly(dA) as a template was investigated. Both dTMP and dTTP were shown to be the minimal primers of DNA polymerase .alpha., the affinity and V increasing 1.8- and 1,4-fold respectively upon lengthening the primer by each unit from dTMP to d(Tp)9T. Further elongation is accompanied by 1,3-fold affinity enhancement and a decrease in V. For the E. coli enzyme, a similar dependence of affinity of primer d(Tp)4T-d(Tp)14T was observed with the inflexion point corresponding to d(Tp)8T. The individual diastereomers of oligothymidylate ethyl esters (with p'' and p'''' corresponding to enantiomeric configuration) such as d[Tp''(Et)Tp]3Tp''(Et)T, d[Tp''''(Et)Tp]3Tp''''(Et)T, d(Tp)8Tp''(Et)T, d(Tp)8Tp''''(Et)T, d(Tp)8Tp''(Et)TpT, d(Tp)8.cntdot.Tp''''(Et)TpT and completely esterified analogues d[Tp(ET)]7T, d[Tp(Et)]14T were shown to initiate the poly(dA)-dependent polymerization catalyzed by both enzymes. A sum of the obtained results provided the basis for a number of conjectures on the mode of primer and template binding to the enzyme, possible role of their preformed complex, as well as electrostatic interactions and hydrogen bonding.

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