Journal
NATURE COMMUNICATIONS
Volume 11, Issue 1, Pages -Publisher
NATURE RESEARCH
DOI: 10.1038/s41467-020-19165-2
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Funding
- National Institutes of Health (NIH) [GM110387]
- National Science Foundation [MCB-2027902]
- LUMC Fellowship
- Molecular Basis of Disease fellowship from Georgia State University
- Yen Post-Doctoral Fellowship in Interdisciplinary Research
- National Cancer Institute of the National Institutes of Health (NIH) [CAO93577]
- DOE Office of Science User Facility [DE-AC05-00OR22725]
- National Science Foundation XSEDE program [CHE110042]
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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. In high fidelity DNA polymerases the exonuclease site is distal from the polymerization site and it is unknown how the primer strand travels between the two sites when mis-incorporated nucleotides must be removed. Here, the authors perform MD simulations and identify an optimal path for DNA primer strand translocation in the E. coli replicative DNA polymerase III and characterise the kinetics and dynamics of the Pol III pol-to-exo mode transition, which is validated with mutagenesis experiments.
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