Pyrovanadolysis, a Pyrophosphorolysis-like Reaction Mediated by Pyrovanadate, Mn2+, and DNA Polymerase of Bacteriophage T7

Barak Akabayov,Arkadiusz W Kulczyk,Sabine R Akabayov,Christopher Theile,Larry W Mclaughlin,Benjamin Beauchamp,Antoine M Van Oijen,Charles C Richardson

doi:10.1074/jbc.m111.250944

Barak Akabayov, Arkadiusz W Kulczyk + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m111.250944

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Abstract

DNA polymerases catalyze the 3'-5'-pyrophosphorolysis of a DNA primer annealed to a DNA template in the presence of pyrophosphate (PP(i)). In this reversal of the polymerization reaction, deoxynucleotides in DNA are converted to deoxynucleoside 5'-triphosphates. Based on the charge, size, and geometry of the oxygen connecting the two phosphorus atoms of PP(i), a variety of compounds was examined for their ability to carry out a reaction similar to pyrophosphorolysis. We describe a manganese-mediated pyrophosphorolysis-like activity using pyrovanadate (VV) catalyzed by the DNA polymerase of bacteriophage T7. We designate this reaction pyrovanadolysis. X-ray absorption spectroscopy reveals a shorter Mn-V distance of the polymerase-VV complex than the Mn-P distance of the polymerase-PP(i) complex. This structural arrangement at the active site accounts for the enzymatic activation by Mn-VV. We propose that the Mn(2+), larger than Mg(2+), fits the polymerase active site to mediate binding of VV into the active site of the polymerase. Our results may be the first documentation that vanadium can substitute for phosphorus in biological processes.

Highlights

Gene 5 of bacteriophage T7 encodes a DNA polymerase essential for replication of the T7 genome (1)
We show that T7 DNA polymerase catalyzes pyrophosphorolysis and PPi exchange in a manner similar to that found for E. coli DNA polymerase I (29)
If the incoming deoxynucleoside 5Ј-triphosphate (dNTP) is present, there is an exchange of PPi with the ␤,␥-phosphates of the nucleotide

Summary

Introduction

Gene 5 of bacteriophage T7 encodes a DNA polymerase essential for replication of the T7 genome (1). Pyrophosphorolysis is a true reversal of the polymerization reaction in that the products are dNTPs, and it has the same requirements for a template and a 3Ј-hydroxyl-terminated primer (5). Inhibition of pyrophosphatase or transient localization of pyrophosphate near the active site can result in pyrophosphorolysis Such a reversal in the reaction would enable polymerases such as the Y family of DNA polymerases or RNA polymerases lacking a proofreading exonuclease activity to use pyrophosphorolysis as an alternative means of proofreading (7). B, enlargement of the active site of T7 DNA polymerase containing the 3Ј terminus of the primer with the two Mg2ϩ ions (green) and dATP (left) or PPi (right) from an overlay of the two metal ions in the Dpo crystal structure together with aration were

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