Role of the catalytic metal during polymerization by DNA polymerase lambda

Abstract

The incorporation of dNMPs into DNA by polymerases involves a phosphoryl transfer reaction hypothesized to require two divalent metal ions. Here we investigate this hypothesis using as a model human DNA polymerase lambda (Pol lambda), an enzyme suggested to be activated in vivo by manganese. We report the crystal structures of four complexes of human Pol lambda. In a 1.9 A structure of Pol lambda containing a 3'-OH and the non-hydrolyzable analog dUpnpp, a non-catalytic Na+ ion occupies the site for metal A and the ribose of the primer-terminal nucleotide is found in a conformation that positions the acceptor 3'-OH out of line with the alpha-phosphate and the bridging oxygen of the pyrophosphate leaving group. Soaking this crystal in MnCl2 yielded a 2.0 A structure with Mn2+ occupying the site for metal A. In the presence of Mn2+, the conformation of the ribose is C3'-endo and the 3'-oxygen is in line with the leaving oxygen, at a distance from the phosphorus atom of the alpha-phosphate (3.69 A) consistent with and supporting a catalytic mechanism involving two divalent metal ions. Finally, soaking with MnCl2 converted a pre-catalytic Pol lambda/Na+ complex with unreacted dCTP in the active site into a product complex via catalysis in the crystal. These data provide pre- and post-transition state information and outline in a single crystal the pathway for the phosphoryl transfer reaction carried out by DNA polymerases.

Figure 1. Structure of Pol λ in complex with dUpnpp

Stereo view of the Pol λ active site. A Mg²⁺ ion occupies the metal B binding site (green), but a non-catalytic Na⁺ ion is present in the metal A binding site (light blue). As a result, the 3’-OH (black arrow) is located far from the phosphorus of the α-phosphate (see yellow dotted line) and is not in line with this atom and the bridging oxygen (nitrogen in dUpnpp) of the pyrophosphate leaving group. A simulated annealing Fobs-Fcalc omit map is shown (blue) contoured at 4σ. The three catalytic aspartates are shown in gray.

Figure 2. Soaking with Mn²⁺ induces a catalytic conformation

Stereo view of the Pol λ active site. A Mn²⁺ ion (magenta) now occupies the metal A binding site. The 3’-OH is now at a catalytically relevant distance (see yellow dotted line) from the phosphorus of the α-phosphate and both of these atoms are in line with the bridging oxygen (nitrogen in the analog) of the pyrophosphate leaving group. A simulated annealing Fobs-Fcalc omit map contoured at 4σ is shown in blue and an anomalous difference density map contoured at 5σ is shown in magenta. The three catalytic aspartates are shown in gray.

Figure 3. Metal-activated catalysis

A. Pre-catalytic complex of Pol λ with dCTP. Under these crystallization conditions, Na⁺ occupies the metal A binding site (light blue), resulting in a non-catalytic conformation. As a result, the substrate (yellow) is unreacted. A simulated annealing Fobs-Fcalc omit map contoured at 4σ is shown in blue. B. The crystals shown in (A) were soaked in the presence of Mn²⁺ atoms. To clearly observe the effects of the soak, a Fobs(pre)-Fobs(post) difference density map was calculated. This map only expresses the differences between the two datasets (pre- and post-soak). It is contoured at 5σ (light blue) and at -5σ (yellow). Thus, yellow indicates electron density appearing upon soaking the crystal, while light blue indicates electron density that disappeared in response to the soak. Superimposed to the Fobs(pre)-Fobs(post) difference density map is an anomalous difference density map contoured at 8σ (red). This map indicates the position of the Mn²⁺ atoms after the soak.

Figure 4. The inline displacement reaction

An overlay of the Pol λ precatalytic complex with dUpnpp and a post-catalytic complex (PDB 1XSP) reveals the details of the inline displacement reaction. The polymerization reaction results in an inversion in the stereochemical configuration of the phosphorus atom of the α-phosphate group. The line of transfer is shown as a yellow dotted line.

Figure 5. Overlay of the Pol β and Pol λ complexes with dUpnpp

The superimposition is centered on the active site (19 Cα-atoms, Pol λ residues 413 to 431, rmsd 0.251Å). The active site of Pol λ is shown and the equivalent atoms from the Pol β structure are shown transparent in the same colour.