Pyrophosphate release acts as a kinetic checkpoint during high-fidelity DNA replication by the Staphylococcus aureus replicative polymerase PolC

Abstract

Bacterial replication is a fast and accurate process, with the bulk of genome duplication being catalyzed by the α subunit of DNA polymerase III within the bacterial replisome. Structural and biochemical studies have elucidated the overall properties of these polymerases, including how they interact with other components of the replisome, but have only begun to define the enzymatic mechanism of nucleotide incorporation. Using transient-state methods, we have determined the kinetic mechanism of accurate replication by PolC, the replicative polymerase from the Gram-positive pathogen Staphylococcus aureus. Remarkably, PolC can recognize the presence of the next correct nucleotide prior to completing the addition of the current nucleotide. By modulating the rate of pyrophosphate byproduct release, PolC can tune the speed of DNA synthesis in response to the concentration of the next incoming nucleotide. The kinetic mechanism described here would allow PolC to perform high fidelity replication in response to diverse cellular environments.

An NTP-driven mechanism for the nucleotide addition cycle of Escherichia coli RNA polymerase during transcription

“…In both studies, the results were interpreted to indicate that pyrophosphate release is the rate limiting step during processive transcription [ 45 , 52 ]. In the case of Staphylococcus aureus replicative polymerase PolC, the rate of pyrophosphate release is slow relative to nucleotide incorporation during one round of the NAC [ 53 ]. However, unlike in the case of RNA polymerase, the binding of subsequent nucleotides for incorporation eliminates the slow release of pyrophosphate.…”

Transient State Kinetics of Plasmodium falciparum Apicoplast DNA Polymerase Suggests the Involvement of Accessory Factors for Efficient and Accurate DNA Synthesis

“…As a part of the replisome, a DNA polymerase interacts with various components of the replication machinery. Nearly all replicative polymerases interact with a partner protein called the processivity factor, which increases the processivity of a polymerase either by reducing the dissociation rate of the prechemistry binary complex or by increasing the rate of chemistry. , In certain cases, a processivity factor may even increase the polymerase’s affinity for nucleotides . However, to date, no processivity factor for apPol has been identified, and it has been proposed that the relatively small size of the apicoplast genome (∼35 kb) might allow apPol to perform replicative synthesis without an associated factor .…”

“…The structural information has been complemented with preliminary functional characterizations of apPol using steady-state multiple-turnover kinetics. 8,14,15 All DNA polymerases, with a few exceptions, 16,17 follow the same sequence of events when incorporating a deoxynucleoside triphosphate (dNTP) into a growing DNA strand (Figure 1B). Different polymerases achieve their unique catalytic signatures, required for their specialized functions, by virtue of altered rate constants governing the individual steps of the enzymatic cycle.…”

Pre-Steady-State Kinetic Characterization of an Antibiotic-Resistant Mutation of Staphylococcus aureus DNA Polymerase PolC

“…PolC concentration was determined by UV absorbance at 280 nm using a theoretical extinction coefficient of 113,000 M -1 cm -1 . β-clamp was purified as described previously (17).…”

“…To effectively target the different reaction intermediates, a comprehensive kinetic description of these states is essential and can be achieved using transient-state rather than steady-state kinetic measurements. We have recently determined the mechanism S. aureus PolC using transient-state kinetics (16, 17), the first detailed kinetic characterization of any C-family DNA polymerase. The structure of a thermophilic homolog of S. aureus PolC in a ternary complex that is poised for catalysis (Fig.…”