Reduced structural flexibility for an exonuclease deficient DNA polymerase III mutant

Abstract

DNA synthesis, carried out by DNA polymerases, requires balancing speed and accuracy for faithful replication of the genome. High fidelity DNA polymerases contain a 3′–5′ exonuclease domain that can remove misincorporated nucleotides on the 3′ end of the primer strand, a process called proofreading. The E. coli replicative polymerase, DNA polymerase III, has spatially separated (~55 Å apart) polymerase and exonuclease subunits. Here, we report on the dynamics of E. coli DNA polymerase III proofreading in the presence of its processivity factor, the β2-sliding clamp, at varying base pair termini using single-molecule FRET. We find that the binding kinetics do not depend on the base identity at the termini, indicating a tolerance for DNA mismatches. Further, our single-molecule data and MD simulations show two previously unobserved features: (1) DNA Polymerase III is a highly dynamic protein that adopts multiple conformational states while bound to DNA with matched or mismatched ends, and (2) an exonuclease-deficient DNA polymerase III has reduced conformational flexibility. Overall, our single-molecule experiments provide high time-resolution insight into a mechanism that ensures high fidelity DNA replication to maintain genome integrity.

Polymerization and editing modes of a high-fidelity DNA polymerase are linked by a well-defined path

“…Pol III thumb domain residues Y453 and K461 stabilize the separated primer. The importance of Y453 was noted in previous experimental studies 7 , 43 . We also noted a loop in the thumb domain (P464-M469) that protrudes into the DNA major groove in polymerization mode, while directly binding the template strand in exonuclease mode.…”

Quantitative comparison between sub-millisecond time resolution single-molecule FRET measurements and 10-second molecular simulations of a biosensor protein

“…Single-molecule Förster Resonance Energy Transfer (smFRET), an experimental technique used to study the dynamics of a wide variety of biomolecules, from polymerases to ribosomes, to G-protein coupled receptors, could benefit greatly from quantitative MD simulation comparison [47][48][49][50][51]. Investigations involving MD interpretations of smFRET experiments have been a major focus for some time [52].…”

Error-Speed Correlations in Biopolymer Synthesis

“…These correlations provide a way to identify the error correction mechanism adopted by an enzyme from experimental data. This approach can circumvent the characterization of these enzymes by measuring all kinetic rates of the underlying reaction network [8][9][10][11][12][13][14][15].We consider an enzyme that replicates an existing template polymer by sequentially incorporating monomers into a copy polymer (Figure 1a). In a given time interval T , the enzyme synthesizes a copy made up of a number of monomers L. Because of thermal fluctuations, enzymes sometimes incorporate wrong monomers (w) that do not match the template, instead of the right ones (r).…”

“…These correlations provide a way to identify the error correction mechanism adopted by an enzyme from experimental data. This approach can circumvent the characterization of these enzymes by measuring all kinetic rates of the underlying reaction network [8][9][10][11][12][13][14][15].…”