Hydrogen‐Bonding Capability of Nonpolar Difluorotoluene Nucleoside in Replication Complexes

Nucleoside analogs are used as chemotherapeutic agents as well as specific probes in chemogenetics to define structure‐function relationships of nucleic acids at an atomic level. The introduction of difluorotoluene as a thymine isostere (dF) by Kool et al. challenged the Watson‐Crick (W‐C) dogma that hydrogen‐bonds (H‐bonds) between complimentary bases (A/T or G/C) are an absolute requirement for accurate DNA replication. Results with dF and other isosteres prompted Kool et al. to hypothesize that the shape of nascent base‐pairs was a more important factor for base discrimination than inter‐base W‐C H‐bonds. Their hypothesis remains controversial because the underlying assumptions have never been validated by results from relevant replication complexes. Here we report high‐resolution crystal structures and kinetic behavior of RB69 DNA polymerase (RB69pol) Y567A/L561A/S565G triple mutant ternary complexes with a templating dF opposite all four incoming dNTPs (dATP, dCTP, dGTP & dTTP). We show that: ( i ) dF has unexpected H‐bonding capability; ( ii ) the shape of nascent base‐pairs is distorted from canonic W‐C nascent base‐pairs and; ( iii) the shapes of dATP/dF and dGTP/dF pairs differ from dA/dF and dG/dF pairs when they are embedded in duplex DNA. These findings provide an explanation for the observed selectivity of dF for dATP and strongly support the concept that H‐bonding between incoming dNTPs and a templating base or base analog is crucial for dNTP discrimination. Thus it appears that, although the shape of the nascent base‐pair has a role in nucleotide selectivity, its importance is overshadowed by H‐bonding between the templating base and the incoming dNTP.