Pyrophosphate hydrolysis is an intrinsic and critical step of the DNA synthesis reaction

Abstract

DNA synthesis by DNA polymerases (dPols) is central to duplication and maintenance of the genome in all living organisms. dPols catalyze the formation of a phosphodiester bond between the incoming deoxynucleoside triphosphate and the terminal primer nucleotide with the release of a pyrophosphate (PPi) group. It is believed that formation of the phosphodiester bond is an endergonic reaction and PPi has to be hydrolyzed by accompanying pyrophosphatase enzymes to ensure that the free energy change of the DNA synthesis reaction is negative and it can proceed in the forward direction. The fact that DNA synthesis proceeds in vitro in the absence of pyrophosphatases represents a long-standing conundrum regarding the thermodynamics of the DNA synthesis reaction. Using time-resolved crystallography, we show that hydrolysis of PPi is an intrinsic and critical step of the DNA synthesis reaction catalyzed by dPols. The hydrolysis of PPi occurs after the formation of the phosphodiester bond and ensures that the DNA synthesis reaction is energetically favorable without the need for additional enzymes. Also, we observe that DNA synthesis is a two Mg2+ ion assisted stepwise associative SN2 reaction. Overall, this study provides deep temporal insight regarding the primary enzymatic reaction responsible for genome duplication.

Visualizing Rev1 Catalyze Protein-template DNA Synthesis

“…Therefore, the breakdown of pyrophosphate by Rev1 could explain why a third metal is not seen during Rev1-mediated insertion. This is consistent with DNA Pol IV, which also did not contain a third metal in the product state for Mg 2+ -facilitated insertion (30). However, it is possible that pyrophosphate hydrolysis leads to a short-lived state containing the third product metal that we were unable to capture in the structural snapshots presented here.…”

“…2C). This indicates hydrolysis of pyrophosphate occurred in the Rev1 active site following dCMP insertion, which is consistent with recent time-resolved crystallography with DNA Pol IV(30). The two monophosphates in the Rev1 active site are no longer bound by a nucleotide associated metal and subtle structural changes occur to alleviate the electrostatic repulsion from the negatively charged species within the Rev1 active site.…”

“…The first evidence of pyrophosphate hydrolysis in the active site of a DNA polymerase was recently described for DNA Pol IV (30). Our data indicates Rev1 also breakdowns pyrophosphate in the active site in crystallo and in solution.…”

“…Our data indicates Rev1 also breakdowns pyrophosphate in the active site in crystallo and in solution. Pyrophosphate hydrolysis in the active site of a DNA polymerase is proposed to make the DNA synthesis reaction energetically favorable (30). In the case of Rev1, it is likely important for driving the forward reaction in two ways.…”

Vitamin B12 May Inhibit RNA-Dependent-RNA Polymerase Activity of nsp12 from the SARS-CoV-2 Virus

“…The RNA and incoming nucleotide of the transformed coordinates of the ternary complex of Q-beta replicase (3AVX) were transferred onto the homology model of SCV2-nsp12 (11). The local structure in the active site was altered manually to ensure co-ordination of the co-factor ion by the catalytic residues and the triphosphate moiety of the incoming nucleotide as seen previously for DNA polymerases (12). The structure prepared in this way was subjected to energy minimization using DESMOND module of the Schrödinger suite.…”