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

The emergence and spread of antibiotic resistance in bacterial pathogens are serious and ongoing threats to public health. Since chromosome replication is essential to cell growth and pathogenesis, the essential DNA polymerases in bacteria have long been targets of antimicrobial development, although none have yet advanced to the market. Here, we use transient-state kinetic methods to characterize the inhibition of the PolC replicative DNA polymerase from Staphylococcus aureus by 2-methoxyethyl-6-(3'-ethyl-4'-methylanilino)uracil (ME-EMAU), a member of the 6-anilinouracil compounds that specifically target PolC enzymes, which are found in low-GC content Gram-positive bacteria. We find that ME-EMAU binds to S. aureus PolC with a dissociation constant of 14 nM, more than 200-fold tighter than the previously reported inhibition constant, which was determined using steady-state kinetic methods. This tight binding is driven by a very slow off rate of 0.006 s-1. We also characterized the kinetics of nucleotide incorporation by PolC containing a mutation of phenylalanine 1261 to leucine (F1261L). The F1261L mutation decreases ME-EMAU binding affinity by at least 3,500-fold but also decreases the maximal rate of nucleotide incorporation by 11.5-fold. This suggests that bacteria acquiring this mutation would be likely to replicate slowly and be unable to out-compete wild-type strains in the absence of inhibitors, reducing the likelihood of the resistant bacteria propagating and spreading resistance.

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

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.