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.

Substrate specificity and proposed structure of the proofreading complex of T7 DNA polymerase

“…We emphasize that our structural model may have some limitations in terms of precise atomic interactions but nonetheless, the model provides reasonable constraints on how the DNA interacts with the enzyme in the exonuclease active site. As shown in Figure 4 , C and D , three bases must melt from the DNA duplex for the primer to fully partition into the exonuclease active site, and this is consistent with estimates from homologous enzymes ( 42 , 46 ). This preliminary pathway from the linear interpolation between the two active sites gives a structural model for the conformational dynamics underlying the movement of the DNA from the pol to the exo site.…”

“…Our model reveals significant structural changes in the enzyme when the DNA primer strand transfers from the pol site into the exonuclease active site. Similar to a recent study on E. coli DNA polymerase III that used targeted MD simulations to generate the minimum energy path between the exonuclease and polymerase complexes (47), our model suggests that the DNA backtracks. Rotation of the thumb domain and the thioredoxin-binding domain that interacts with downstream DNA serves to unwind the DNA.…”

Structural dynamics and determinants of 2-aminoadenine specificity in DNA polymerase DpoZ of vibriophage ϕVC8

“…The feature that one should look for is subtle and dynamical in nature, probably involving an allosteric network of residues participating in the transition from the elongation (polymerase) mode of the enzyme to the proofreading (exonuclease) one. This communication pathway has been the subject of active research and is still unresolved and being investigated ( 63 ); it might actually be somewhat different in different polymerase families. A possible suggestion (that could be verified by protein engineering) concerns the ϕVC8 DpoZ insertion 117–127, which corresponds to the region 102–122 in T7 PolA, assumed to be implicated in DNA transfer between pol and exo sites ( 47 ).…”

Mechanism of Rad26-assisted rescue of stalled RNA polymerase II in transcription-coupled repair

“…To discover Pol II–Rad26 allosteric residue networks and communication mechanisms, we applied graph-theoretical approaches 33 , 49 , 50 that map dynamic information from our extensive MD simulations onto graphs representing the protein topology (i.e. residues are represented by graph nodes; edges connect contacting residues).…”