Enzymatic synthesis of deoxyribonucleic acid.

SUMMARY The pyrophosphate (PPi) exchange reaction catalyzed by Escherichia cob DNA polymerase is identical with the polymerization reaction in its requirements for a template, strict specificity in base pairing, and a 3’-hydroxyl-terminated primer strand. However, in contrast to polymerization, appreciable exchange is obtained in the absence of a full complement of deoxyribonucleoside triphosphates. lnhibition of synthesis by PPi as measured by the difference between PPi release and nucleotide incorporation from deoxyribonucleoside triphosphates, can be accounted for by PPi exchange. The PPi exchange reaction appears to represent the removal by PPi of the newly incorporated nucleotide prior to its complete stabilization by the entry of the next triphosphate. The degradation of DNA by PPi (pyrophosphorolysis) appears to differ from the PPi exchange reaction in that it is inhibited by deoxyribonucleoside triphosphates, attains a steady state plateau, and has a lower pH optimum and a slower rate. Some of these differences may depend on the requirement that progressive pyrophosphorolysis places on progressive movement of the DNA chain relative to the enzyme. The results are discussed in relation to the mechanism of the PPi exchange reaction and to all polymerase functions in a model with a single active center for the enzyme.

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