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 broa d host range plasmid RSF1010 harbours a gene (repA) encoding for one o f the smallest known oligomeric helicases, RepA, a homo-hexamer with 3 0kDa subunits. Electron micrographs indicate that the overall shape of RepA resembles a hexagon with globular monomers at the corners, diame ter 140 Angstrom, and a central channel. Below pH 6, the molecules agg regate into tubular structures. The enzyme has been purified and cryst allized using the hanging-drop vapour-diffusion method with polyethyle neglycol monomethylether as precipitating agent. The crystals exhibit the monoclinic space group P2(1) with unit-cell parameters a = 105.8, b = 180.3, c = 115.4 Angstrom, beta = 95.2 degrees, and diffract to 3. 5 Angstrom resolution using rotating-anode Cu K alpha radiation. Assum ing two 180kDa molecules per asymmetric unit, the volume per unit weig ht is V-m = 3.06 Angstrom(3) Da(-1), equivalent to a solvent content o f 60%. A self-rotation search indicates that the sixfold axis of the h examer is parallel to the ac plane and inclined at about 2 degrees to the c axis. The two hexamers are oriented head-to-head with point-grou p symmetry D-6.