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

Dodd, Thomas; Botto, Margherita; Paul, Fabian; Fernandez-Leiro, Rafael; Lamers, Meindert H.; Ivanov, Ivaylo

doi:10.1038/s41467-020-19165-2

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

Journal Article · Fri Oct 23 00:00:00 EDT 2020 · Nature Communications

DOI:https://doi.org/10.1038/s41467-020-19165-2· OSTI ID:1816482

^[1]; Botto, Margherita ^[2]; Paul, Fabian ^[3]; ^[4]; ^[2]; ^[5]

Georgia State Univ., Atlanta, GA (United States). Dept. of Chemistry; Georgia State Univ., Atlanta, GA (United States). Center for Diagnostics and Therapeutics; OSTI
Leiden Univ. (Netherlands). Leiden Univ. Medical Center. Dept. of Cell and Chemical Biology
Univ. of Chicago, IL (United States). Dept. of Biochemistry & Molecular Biology
Spanish National Cancer Research Centre (CNIO), Madrid (Spain)
Georgia State Univ., Atlanta, GA (United States). Dept. of Chemistry; Georgia State Univ., Atlanta, GA (United States). Center for Diagnostics and Therapeutics

Proofreading by replicative DNA polymerases is a fundamental mechanism ensuring DNA replication fidelity. In proofreading, mis-incorporated nucleotides are excised through the 3'-5' exonuclease activity of the DNA polymerase holoenzyme. The exonuclease site is distal from the polymerization site, imposing stringent structural and kinetic requirements for efficient primer strand transfer. Yet, the molecular mechanism of this transfer is not known. Here we employ molecular simulations using recent cryo-EM structures and biochemical analyses to delineate an optimal free energy path connecting the polymerization and exonuclease states of E. coli replicative DNA polymerase Pol III. We identify structures for all intermediates, in which the transitioning primer strand is stabilized by conserved Pol III residues along the fingers, thumb and exonuclease domains. We demonstrate switching kinetics on a tens of milliseconds timescale and unveil a complete pol-to-exo switching mechanism, validated by targeted mutational experiments.

View Accepted Manuscript (DOE)

Research Organization:: Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities Division

Grant/Contract Number:: AC05-00OR22725

OSTI ID:: 1816482

Journal Information:: Nature Communications, Journal Name: Nature Communications Journal Issue: 1 Vol. 11; ISSN 2041-1723

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: English

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