Search for a command to run...
Fidelity of DNA polymerases, defined as their ability to incorporate a correct dNMP opposite an undamaged template nucleotide, is a key factor in ensuring genome stability. Many DNA polymerases have been extensively investigated to understand the structural and biochemical basis of their fidelity. However, the great majority of these studies employed simple primer-template substrates, disregarding the possible effects of more complex DNA structures. Here, we have estimated the fidelity of a single dNMP incorporation by the Klenow fragment of <i>Escherichia coli</i> DNA polymerase I (KF) and bacteriophage RB69 DNA polymerase (RBpol) during the strand displacement synthesis, a situation common in lagging strand replication and DNA repair. Although KF was more efficient than RBpol in the presence of a downstream strand, both polymerases demonstrated a 3-fold (up to an order of magnitude) increase in the fidelity compared with the primer-template system. To assess the dependence of fidelity on the energy spent on the disruption of the base pair ahead, we varied its strength using mismatches and synthetic base analogs. Both KF and RBpol generally traded efficiency for fidelity, making fewer errors when they had to disrupt stronger base pairs. Thus, the energetic penalty imposed by the downstream strand acts as a fidelity checkpoint, enhancing discrimination against incorrect nucleotides.