Structural insights into DNA replication without hydrogen bonds

Abstract

The genetic alphabet is composed of two base pairs, and the development of a third, unnatural base pair would increase the genetic and chemical potential of DNA. d5SICS-dNaM is one of the most efficiently replicated unnatural base pairs identified to date, but its pairing is mediated by only hydrophobic and packing forces, and in free duplex DNA it forms a cross-strand intercalated structure that makes its efficient replication difficult to understand. Recent studies of the KlenTaq DNA polymerase revealed that the insertion of d5SICSTP opposite dNaM proceeds via a mutually induced-fit mechanism, where the presence of the triphosphate induces the polymerase to form the catalytically competent closed structure, which in turn induces the pairing nucleotides of the developing unnatural base pair to adopt a planar Watson-Crick-like structure. To understand the remaining steps of replication, we now report the characterization of the prechemistry complexes corresponding to the insertion of dNaMTP opposite d5SICS, as well as multiple postchemistry complexes in which the already formed unnatural base pair is positioned at the postinsertion site. Unlike with the insertion of d5SICSTP opposite dNaM, addition of dNaMTP does not fully induce the formation of the catalytically competent closed state. The data also reveal that once synthesized and translocated to the postinsertion position, the unnatural nucleobases again intercalate. Two modes of intercalation are observed, depending on the nature of the flanking nucleotides, and are each stabilized by different interactions with the polymerase, and each appear to reduce the affinity with which the next correct triphosphate binds. Thus, continued primer extension is limited by deintercalation and rearrangements with the polymerase active site that are required to populate the catalytically active, triphosphate bound conformation.

Figure 1

The d5SICS-dNaM unnatural base pair, with a natural Watson-Crick base pair shown for comparison.

Figure 2

Open binary and pre-catalytic ternary complex of KTQ_d5SICS and KTQ_{d5SICS-dNaMTP}, respectively. (A) The natural base pair at the post-insertion site, the templating d5SICS, and Tyr671 are shown as sticks and the O- and N-helices are shown as cartoon. Simulated annealing mFo-DFc omit map around d5SICS is shown, contoured at 3σ. (B) Same arrangement as in (A) but for the ternary complex KTQ_{d5SICS-dNaMTP}. Simulated annealing mFo-DFc omit map around the bound dNaMTP and the coordinated Mg²⁺ ion (green sphere) and associated water molecules (red spheres) is shown, contoured at 3σ. (C) Superposition of KTQ_d5SICS (cyan), KTQ_{d5SICS-dNaMTP} (orange) and KTQ_{dNaM-d5SICSTP} (PDB ID 3SZ2, purple) shows the open, partially closed and closed state of the enzyme.

Figure 3

Primer termini of open binary complexes with dNaM-d5SICS in the post-insertion site. KTQ(E1)_dNaM-d5SICS, KTQ(E2)_dNaM-d5SICS, and KTQ(E2)_d5SICS-dNaM are labeled and shown in green, blue, and red, respectively. The intercalated unnatural base pair is shown in dark green, dark blue and pink, respectively, surrounded by their simulated annealing mFo-DFc omit maps contoured at 3σ. C1’-C1’ distances (Å) within each unnatural pair are shown.

Figure 4

Comparison of KTQ(E1)_dNaM-d5SICS (green) and KTQ(E2)_dNaM-d5SICS (blue) with KTQ_dG with a natural dC-ddG base pair at the post-insertion site (grey). (A) Superposition of duplex portion of primer/template of KTQ(E1)_dNaM-d5SICS and KTQ(E2)_dNaM-d5SICS with KTQ_dG. The unnatural base pair in the post-insertion site is shown in ball and stick representation. (B) Superposition of KTQ(E1)_dNaM-d5SICS and KTQ_dG, shown as cartoon. The finger and palm domains which are only slightly affected by the unnatural base pair are transparent. The larger movement of the thumb domain is indicated with black arrows. (C and D) Superposition of unnatural and natural base pair (from KTQ_dG) at post-insertion site with distance between C1’ atoms indicated in Å, (C) KTQ(E1)_dNaM-d5SICS and (D) KTQ(E2)_dNaM-d5SICS.

Figure 5

Proposed mechanism of replication. Intermediates not yet validated by structural studies (i.e. extension complexes) are shown in lighter color. The steps corresponding to incorporation of the unnatural triphosphate and subsequent extension of the nascent unnatural base pair are indicated. The O-helix of the protein is shown, phosphates are indicated with open circles, natural nucleotides are indicated with open rectangles, and the unnatural nucleotides are indicated with grey and black rectangles.