Insights into the Replisome from the Structure of a Ternary Complex of the DNA Polymerase III α-Subunit

Wing, Richard A.; Bailey, Scott; Steitz, Thomas A.

doi:10.1142/9789811215865_0041

Cited by 2 publications

(3 citation statements)

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“…Compared to viral and bacteriophage replicative DNA polymerases, mechanistic studies of cellular replicative polymerases have lagged behind. Among the cellular replicative polymerases, the bacterial C-family polymerases have been particularly challenging to study, largely because of weak binding to DNA and the unique catalytic cycle of the enzyme, but the available structures of several essential C-family polymerases (7,8,(31)(32)(33)(34)(35)(36) and our transientstate kinetic characterization of S. aureus PolC (16,17,24) provide the framework needed for a mechanistic structure-function approach to developing antibiotics targeting bacterial DNA replication.…”

Section: Discussionmentioning

confidence: 99%

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

Nelson-Rigg

Fagan

Jaremko

et al. 2022

Preprint

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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 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, 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 3500-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 inhibitor, reducing the likelihood of the resistant bacteria propagating and spreading resistance.

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Section: Discussionmentioning

confidence: 99%

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

Nelson-Rigg

Fagan

Jaremko

et al. 2022

Preprint

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“…Thermus aquaticus (Taq) alpha (PDB ID 3E0D) is a homologue of Eco Pol III alpha; PolC (PDB ID 3F2B) is a replicative C family polymerase from Gram-positive Geobacillus kaustophilus (Gka). 28,35,36 The active site residues in the Eco and Taq structures are identical as they share an overall 40% sequence identity. Gka PolC belongs to a different class of C family polymerases that are found in low G +C Gram-positive bacteria and shares low sequence identity of 25% to Eco alpha.…”

Section: ■ Discussionmentioning

confidence: 99%

“…The three structures available for C family polymerases, 2HNH, 3E0D, and 3F2B, were aligned using the Matchmaker tool in UCSF Chimera software. 28,35,36,41 The local active site of 2HNH was manually aligned with the active site of human DNA polymerase β (PDB ID 1BPX) using Pymol software. 42−44…”

Section: Biochemistrymentioning

confidence: 99%

Prediction of Active Site and Distal Residues in E. coli DNA Polymerase III alpha Polymerase Activity

et al. 2018

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The process of DNA replication is carried out with high efficiency and accuracy by DNA polymerases. The replicative polymerase in E. coli is DNA Pol III, which is a complex of 10 different subunits that coordinates simultaneous replication on the leading and lagging strands. The 1160-residue Pol III alpha subunit is responsible for the polymerase activity and copies DNA accurately, making one error per 10 nucleotide incorporations. The goal of this research is to determine the residues that contribute to the activity of the polymerase subunit. Homology modeling and the computational methods of THEMATICS and POOL were used to predict functionally important amino acid residues through their computed chemical properties. Site-directed mutagenesis and biochemical assays were used to validate these predictions. Primer extension, steady-state single-nucleotide incorporation kinetics, and thermal denaturation assays were performed to understand the contribution of these residues to the function of the polymerase. This work shows that the top 15 residues predicted by POOL, a set that includes the three previously known catalytic aspartate residues, seven remote residues, plus five previously unexplored first-layer residues, are important for function. Six previously unidentified residues, R362, D405, K553, Y686, E688, and H760, are each essential to Pol III activity; three additional residues, Y340, R390, and K758, play important roles in activity.

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Insights into the Replisome from the Structure of a Ternary Complex of the DNA Polymerase III α-Subunit

Cited by 2 publications

References 0 publications

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

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

Prediction of Active Site and Distal Residues in E. coli DNA Polymerase III alpha Polymerase Activity

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