Polymerization and editing modes of a high-fidelity DNA polymerase are linked by a well-defined path
Abstract
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.
- Authors:
-
[1];
[4];
[2];
[1]
- Georgia State Univ., Atlanta, GA (United States). Dept. of Chemistry; Georgia State Univ., Atlanta, GA (United States). Center for Diagnostics and Therapeutics
- 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)
- Publication Date:
- Research Org.:
- Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities Division
- OSTI Identifier:
- 1816482
- Grant/Contract Number:
- AC05-00OR22725
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Nature Communications
- Additional Journal Information:
- Journal Volume: 11; Journal Issue: 1; Journal ID: ISSN 2041-1723
- Publisher:
- Nature Publishing Group
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES
Citation Formats
Dodd, Thomas, Botto, Margherita, Paul, Fabian, Fernandez-Leiro, Rafael, Lamers, Meindert H., and Ivanov, Ivaylo. Polymerization and editing modes of a high-fidelity DNA polymerase are linked by a well-defined path. United States: N. p., 2020.
Web. doi:10.1038/s41467-020-19165-2.
Dodd, Thomas, Botto, Margherita, Paul, Fabian, Fernandez-Leiro, Rafael, Lamers, Meindert H., & Ivanov, Ivaylo. Polymerization and editing modes of a high-fidelity DNA polymerase are linked by a well-defined path. United States. https://doi.org/10.1038/s41467-020-19165-2
Dodd, Thomas, Botto, Margherita, Paul, Fabian, Fernandez-Leiro, Rafael, Lamers, Meindert H., and Ivanov, Ivaylo. Fri .
"Polymerization and editing modes of a high-fidelity DNA polymerase are linked by a well-defined path". United States. https://doi.org/10.1038/s41467-020-19165-2. https://www.osti.gov/servlets/purl/1816482.
@article{osti_1816482,
title = {Polymerization and editing modes of a high-fidelity DNA polymerase are linked by a well-defined path},
author = {Dodd, Thomas and Botto, Margherita and Paul, Fabian and Fernandez-Leiro, Rafael and Lamers, Meindert H. and Ivanov, Ivaylo},
abstractNote = {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.},
doi = {10.1038/s41467-020-19165-2},
journal = {Nature Communications},
number = 1,
volume = 11,
place = {United States},
year = {Fri Oct 23 00:00:00 EDT 2020},
month = {Fri Oct 23 00:00:00 EDT 2020}
}
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