Fig. 6: Insights into discrimination from site-directed mutagenesis.
From: Watching a double strand break repair polymerase insert a pro-mutagenic oxidized nucleotide
Catalytic efficiencies (kcat/KM, in μM−1 min–1) for a dGTP (see Supplementary Table 7) and b 8-oxodGTP (see Supplementary Table 8) insertion by pol μ active site variants opposite Ct (blue line) or At (red line) determined in the presence of Mg2+ (left panel) or Mn2+ (right panel). The error bars represent standard errors (S.E.) from triplicate measurements. c Active site of the Ca2+-bound 8-oxodGTP(syn):Ct(anti) ground state ternary complex (PDB id 7KTJ) of the Lys438Asp variant after a 120 min soak of pol μ/Lys438Asp-DNA binary complex crystals in a cryo solution containing 2 mM 8-oxodGTP and 20 mM CaCl2. The Lys438Asp active site of the 8-oxodGTP(anti):Ct(anti) insertion after a 120 min soak in 20 mM CaCl2/2 mM 8-oxodGTP and transfer to a cryo solution containing 50 mM MnCl2 for d 30 min (reaction state, PDB id 7KTM), and e 90 min (product state, PDB id 7KTL). Hydrogen bonding between Asp438 and a water molecule coordinating Mnp is shown with a red dashed line. f Active site of the Mg2+-bound 8-oxodGTP(syn):Ct(anti) ternary complex of the Lys438Asp variant (PDB id 7KTK) after soaking as in e and f but instead of the MnCl2 soak, ground state Ca2+-bound ternary complex crystals were soaked in a cryo solution containing 50 mM MgCl2 for 90 min. In the above panels (c–f), simulated annealing Fo–Fc density (green mesh) shown is contoured at a contour level of 3.0 σ, carve radius 2.0 Å, while anomalous density (magenta surface) is shown at 5 σ.
