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.
