KlenTaq polymerase replicates unnatural base pairs by inducing a Watson-Crick geometry

Abstract

Many candidate unnatural DNA base pairs have been developed, but some of the best-replicated pairs adopt intercalated structures in free DNA that are difficult to reconcile with known mechanisms of polymerase recognition. Here we present crystal structures of KlenTaq DNA polymerase at different stages of replication for one such pair, dNaM-d5SICS, and show that efficient replication results from the polymerase itself, inducing the required natural-like structure.

Figure 1. KlenTaq polymerase induces the dNaM-d5SICS unnatural base pair to adopt a natural, Watson-Crick-like structure

Structure of (a) a natural dG-dC base pair and (b) dNaM-d5SICS. Chemical structures are shown at the top of each panel, with a comparison of the structure formed between two nucleotides in duplex DNA (left) and between the templating nucleotide and the incoming triphosphate in the active site of KlenTaq polymerase (right). C1′-C1′ distances are indicated, and CPK renderings are viewed from above and from the minor groove. Only nucleobases are shown with sugar and phosphates omitted for clarity.

Fig. 2. Unnatural base pair formation induces conformational transitions of KlenTaq and the formation of a natural-like ternary complex

Structure of complexes showing helices O and O1, primer-template, and d5SICSTP (if present) in: (a) KTQ_dNaM binary complex (yellow); (b) KTQ_dNaM-d5SICSTP ternary complex (purple); and (c) their superposition highlighting the structural transition induced by d5SICSTP binding. Schematic illustration of conformational transition induced by d5SICSTP binding (d) and superposition of binary and ternary complexes (e). Superposition of KTQ_{dNaM-d5SICSTP} (purple) and KTQ_dG-dCTP (grey) illustrating the similarities of: (f) helices O and O1, and primer-template; (g) active site; and (h) catalytically critical network of side chains, water molecules, and Mg²⁺ ions (water molecules and magnesium ions are shown as light pink and purple spheres, respectively, for KTQ_dNaM-d5SICSTP and as dark grey and light grey spheres, respectively, for KTQ_dG-dCTP; incoming triphosphate is labeled dNTP, templating nucleotide is labeled with dN and dideoxynucleotide at the primer terminus is labeled with ddN, as appropriate).