Requirement for transient metal ions revealed through computational analysis for DNA polymerase going in reverse.

DNA polymerases use a general two-metal ion mechanism for DNA synthesis. Recent time-lapse crystallographic studies identified additional adjunct metal ions in the polymerase active site. One of these ions correlates with appearance of pyrophosphate and was proposed to be involved in pyrophosphorolysis (reverse reaction of DNA synthesis). Because DNA polymerases can use pyrophosphorolysis to remove chain-terminating nucleotides during chemotherapies, a better understanding of this reaction is warranted. Through site-directed mutagenesis, pyrophosphorolysis measurements, and computational analysis, we examine the role of metal ions in the reverse reaction. The results indicate that the product-associated metal ion facilitates pyrophosphorolysis during the early stages of the reaction but deters the reaction at later stages, suggesting dynamic metal behavior that can modulate the chemical equilibrium.