Figure 5. Observing the reverse reaction by time-lapse crystallography.

The pol β active site is shown with key residues indicated; all F_o –F_c omit maps are contoured at 3σ (green). Metal coordination and key distances (Å) are indicated with dashed lines. The carbons of the terminal base pair of the nicked DNA are yellow. The carbons of the upstream DNA are gray. The primer nucleotide upstream of the primer terminus (P10), as well as PNP are indicated. The bridging nitrogen of PNP is colored blue. (a) The active site for the ground state nicked DNA substrate complex with PNP and Ca²⁺ (orange) is shown. (b) An overlay of the substrate nicked DNA/PNP/Ca²⁺ complex (yellow carbons) and the nicked DNA/PP_i/Mn²⁺ product complex (PDB code 4KLH; light blue carbons) is shown. The manganese atom from the PP_i complex is purple. (c) A close-up of the PP_i and PNP phosphate groups from b. The arrows indicate the phosphate oxygen shift for PNP relative to PP_i. The distance between the phosphate and the attacking oxygen for PNP and PP_i is indicated with a dashed line. (d) The reactant complex for the reverse reaction is shown following a short MgCl₂ soak. The Mg²⁺ and water ions are shown as red and blue spheres, respectively. The distances between the bridging water, Arg183, and the nitrogen of PNP are indicated. The catalytic and nucleotide binding metals are labeled as Mg_c and Mg_n, respectively. (e) The final one-nucleotide gapped DNA/dCMPPNP ternary complex is shown following the reverse reaction.