Escherichia coli DnaE Polymerase Couples Pyrophosphatase Activity to DNA Replication

Abstract

DNA Polymerases generate pyrophosphate every time they catalyze a step of DNA elongation. This elongation reaction is generally believed as thermodynamically favoured by the hydrolysis of pyrophosphate, catalyzed by inorganic pyrophosphatases. However, the specific action of inorganic pyrophosphatases coupled to DNA replication in vivo was never demonstrated. Here we show that the Polymerase-Histidinol-Phosphatase (PHP) domain of Escherichia coli DNA Polymerase III α subunit features pyrophosphatase activity. We also show that this activity is inhibited by fluoride, as commonly observed for inorganic pyrophosphatases, and we identified 3 amino acids of the PHP active site. Remarkably, E. coli cells expressing variants of these catalytic residues of α subunit feature aberrant phenotypes, poor viability, and are subject to high mutation frequencies. Our findings indicate that DNA Polymerases can couple DNA elongation and pyrophosphate hydrolysis, providing a mechanism for the control of DNA extension rate, and suggest a promising target for novel antibiotics.

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

“…The conversion of PPi to Pi (Δ G = −7 kcal/mol) is essential to render a large overall negative free energy change (Δ G = −6.5 kcal/mol) to the DNA synthesis reaction ( 45 , 46 ). It was previously believed that dPols act in tandem with pyrophosphatase enzymes that cleave the PPi, so that the coupling of the two reactions provides an overall negative free energy change ( 1 , 8 ). It is clear from this study that the PPi hydrolysis step is inherently part of the complete dNTP incorporation reaction catalyzed by dPols and therefore, the DNA synthesis reaction carried out by dPols is energetically favorable without the need of any other enzyme activity.…”

“…The reaction involving incorporation of a nucleotide into the primer through the formation of the phosphodiester bond along with the release of PPi is expected to have a low free energy change ( 1 , 6 , 7 ). To ensure that the reaction moves in the forward direction, it is believed that the PPi moiety is cleaved by an accompanying pyrophosphatase enzyme to render a large negative free energy change (∼7 kcal/mol) to the DNA synthesis reaction ( 1 , 8–10 ). However, DNA synthesis can proceed smoothly in vitro in the absence of any pyrophosphatase enzyme, and there is no satisfactory explanation available for this long-standing thermodynamic conundrum.…”

An array of basic residues is essential for the nucleolytic activity of the PHP domain of bacterial/archaeal PolX DNA polymerases

“…Besides the bacterial/archaeal PolXs, the bacterial replicative DNA polymerases also contain a PHP domain 17 that can be predicted to be either in an active or inactive conformation depending on either the presence or not of the nine catalytic residues 30 . Thus, in those bacterial replicases whose proofreading function resides in the DnaQ-like exonuclease, as in E. coli Pol III, the PHP domain has been suggested to play a structural role, modulating the stability and activity of the polymerase 31 , as well as it has been demonstrated to control the DNA extension rate by pyrophosphate hydrolysis 32,33 . By contrast, in those replicative DNA polymerases lacking the prototypical DnaQ-like exonuclease, as in the replicases from Mycobacterium tuberculosis 30,34 and T. thermophilus 35 , the PHP domain contains the 3′-5′ exonuclease activity that proofreads the misinserted nucleotides.…”

Comparative genomics of the DNA damage‐inducible network in the Patescibacteria

“…The ppaC gene product is an inorganic pyrophosphatase. These enzymes are known to play an important role in DNA polymerization, and their upregulation in the aftermath of DNA damage could contribute to enhance the processivity of DNA polymerases (Lapenta et al, 2016). In contrast, the predicted Microgenomates LexA regulon is substantially larger and encompasses several canonical members of the SOS response ( Fig.…”