The effect of the 3′,5′ thiophosphoryl linkage on the exonuclease activities of T4 polymerase and the Klenow fragment

Abstract

The 3'----5' exonuclease activities of T4 DNA polymerase and the Klenow fragment of Polymerase I towards the phosphoryl and thiophosphoryl 3',5' linkage were examined under comparable conditions of idling-turnover, duplex hydrolysis and turnover during polymerization. With the T4 enzyme there is a negligible effect of thiosubstitution on these activities; with the Klenow fragment there is a greater than one hundred-fold reduction in rate with the thiolinkage for the exonuclease but not polymerization activities. This inability to hydrolyze rapidly the thiophosphoryl linkage extends to the hydrolytic activity of Exonuclease III. The quantitation of the exonuclease activities of these three proteins under various conditions should aid in the successful employment of thiophosphoryl nucleoside triphosphates for their incorporation into DNA.

A Single Mutation in Human Mitochondrial DNA Polymerase Pol γA Affects Both Polymerization and Proofreading Activities of Only the Holoenzyme

“…Interestingly, mismatch removal occurs at a steadily decreasing rate as the number of mismatches reduces from four to one nucleotide. The rate reduction may reflect a requirement for an increasing number of base pairs to be unwound prior to excision (21,22). The rate of hydrolysis of a single mismatch is slowest among mismatches, suggesting that the lowest energy barrier for a primer to partition between the exo and pol sites is for a single mismatch, in agreement with the fluorescence studies of Millar and co-workers (15).…”

Real-Time DNA Sequencing Using Detection of Pyrophosphate Release

“…In addition, by replacing primer was degraded with longer incubations in the absence of nucleotides. An induced-fit binding mech-all dNTPs by dNTPaSs it may be possible to use DNA polymerases with exonuclease activity (25). anism in the polymerization step (21) selects very efficiently for binding of the correct dNTP with a net…”

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

“…In view of the convincing evidence implicating the 3' -*5' exonuclease activity in ensuring fidelity (10)(11)(12)(13), the extent of the dNTP -* dNMP conversion may thus reflect the degree of proofreading accompanying replication by DNA polymerases possessing such an exonuclease activity (14). In the absence of the following complementary dNTP that is required for normal polymerization, the turnover process is exaggerated since the enzyme is constrained to "idle" at the pnimer terminus until depletion of the available dNTP pool is complete (15). Comparative kinetic studies ofthe turnover reaction with the enzyme held in such an idling mode have allowed the evaluation of base misinsertion frequencies, thus demonstrating an important application ofthe idling-turnover process (16).…”