2026-04-29T04:00:29Zhttps://repisalud.isciii.es/rest/oai/request

oai:repisalud.isciii.es:20.500.12105/121822024-11-29T21:13:14Zcom_20.500.12105_2173com_20.500.12105_2051col_20.500.12105_19597

00925njm 22002777a 4500 dc Dodd, Thomas author Botto, Margherita author Paul, Fabian author Lamers, Meindert H author Ivanov, Ivaylo author Fernandez-Leiro, Rafael author Dodd, Thomas author Botto, Margherita author Paul, Fabian author Lamers, Meindert H author Ivanov, Ivaylo author Fernandez-Leiro, Rafael author 2020-11-01 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. Nat Commun.2020;11(1):5379. http://hdl.handle.net/20.500.12105/12182 33097731 10.1038/s41467-020-19165-2 2041-1723 Nature communications Polymerization and editing modes of a high-fidelity DNA polymerase are linked by a well-defined path.