Mechanism of the idling-turnover reaction of the large (Klenow) fragment of Escherichia coli DNA polymerase I

The mechanism of the idling-turnover reaction catalyzed by the large (Klenow) fragment of Escherichia coli DNA polymerase I has been investigated. The reaction cycle involved is one of excision/incorporation, in which the 3' deoxynucleotide residue of the primer DNA strand is partitioned into its 5'-mono- and 5'-triphosphate derivatives, respectively. Mechanistic studies suggest the 5'-monophosphate product is formed in the first step by simple 3'→5' exonucleolytic cleavage. Rapid polymerization follows with the concomitant release of inorganic pyrophosphate. In the second step, the 5'-triphosphate product is generated by a pyrophosphorolysis reaction, which, despite the low concentration of pyrophosphate that has accumulated, occurs at a rate that is comparable with that of the parallel 3'→5' hydrolysis reaction.