Crystallization and preliminary X-ray crystallographic and electron microscopic study of a bacterial DNA helicase (RSF1010 RepA)

Helicases are ATP-driven enzymes essential for DNA unwinding. The broad host range plasmid RSFI010 harbours a gene (repA) encoding for one of the smallest known oligomeric helicases, RepA, a homo-hexamer with 30 kDa subunits. Electron micrographs indicate that the overall shape of RepA resembles a hexagon with globular monomers at the corners, diameter 140 Å, and a central channel. Below pH 6, the molecules aggregate into tubular structures. The enzyme has been purified and crystallized using the hanging-drop vapour-diffusion method with polyethyleneglycol monomethylether as precipitating agent. The crystals exhibit the monoclinic space group P2₁ with unit-cell parameters a = 105.8, b = 180.3, c = 115.4 Å, β = 95.2°, and diffract to 3.5 Å resolution using rotating-anode Cu Kα radiation. Assuming two 180 kDa molecules per asymmetric unit, the volume per unit weight is V_m = 3.06 Å Da⁻¹, equivalent to a solvent content of 60%. A self-rotation search indicates that the sixfold axis of the hexamer is parallel to the ac plane and inclined at about 2° to the c axis. The two hexamers are oriented head-to-head with point-group symmetry D₆.