DNA pol in action

Error-free DNA replication, and thus genomic stability, critically depends on the ability of DNA polymerases to discriminate correct from incorrect dNTPs during base-by-base extension of the complementary strand. This dNTP-substrate discrimination, known as enzyme fidelity, has been extensively studied with the model eukaryotic DNA polymerase pol β. Functional assays combined with crystal structures of ternary complexes of pol β with DNA and a dNTP positioned opposite the template base suggest that a complementary incoming dNTP promotes conformational changes of active site residues that enhance nucleotide incorporation. This 'induced fit' mechanism is supported by kinetic analyses but is not well characterized structurally because substrate analogs that prevent catalysis are traditionally used to capture reaction intermediates for crystallography. Now, Wilson and colleagues provide a rare glimpse of active complexes containing natural substrates. By soaking ternary-complex crystals in MgCl₂ to initiate nucleotide insertion and freezing them at various time points during catalysis, they obtain snapshots of the active site that reveal differential effects of correct or incorrect dNTP incorporation. Among their most striking findings are that pol β maintains its 'closed conformation' after correct but not incorrect dNTP insertion and that pyrophosphate (PPi), a product of the nucleotidyl transfer reaction, and its associated Mg²⁺ remain bound to the active site after phosphodiester-bond formation. In contrast, misincorporation promotes an 'open' conformation and PPi release. Moreover, a transient third metal-binding site that is observed after correct dNTP incorporation is absent from mismatched ternary complexes, though its function is presently unclear. The finding that pol β ternary complexes show substrate-dependent conformational differences both during and after catalysis suggests that active site configuration can be altered by dNTP misincorporation to restrain subsequent chain elongation, thereby enhancing enzyme fidelity. (Cell 154, 157–168, 2013)