Figure 1. Crystal structure and endonuclease activity of PA_N ^ΔLoop.

(A) Schematic of PA_N (magenta) and PA_N ^ΔLoop (green), and location of the 22-residue loop (orange) replaced by a Gly-Gly-Ser linker in the PA_N ^ΔLoop construct. (B) Two orthogonal views of the overlay of the crystal structures of PA_N and PA_N ^ΔLoop, colored as in A. Key active site residues are shown in stick representation, the paired manganese ions are shown as spheres, and the N- and C-termini are labeled. The coordinates for PA_N are from PDB entry 2W69. The atomic coordinates and structure factors for PA_N ^ΔLoop have been deposited in the Protein Data Bank as PDB entry 4E5E. (C) Coomassie-stained SDS-PAGE of PA_N and PA_N ^ΔLoop showing the amount of protein (µM) in a 10 µl endonuclease activity assay reaction. Molecular weight (MW) markers (kD) are shown on the left. (D) Endonuclease activity assay with PA_N and PA_N ^ΔLoop. Single-stranded DNA plasmid M13mp18 was incubated with increasing concentrations (µM) of PA_N or PA_N ^ΔLoop. Reactions products were resolved on a 1.0% agarose gel stained with ethidium bromide. Molecular weight (MW) ladder (kb) is shown on the left.