FIGURE 1.

Purification of recombinant hChlR1; examination of its helicase and ATPase activities. Wild-type and mutant hChlR1 were overexpressed and purified from 293 cells as described under “Experimental Procedures.” A, left, Coomassie-stained SDS-polyacrylamide gel of purified proteins (4 and 2 μg of hChlR1 and KRm, respectively); right, Western blot analysis of SDS-polyacrylamide gel using affinity-purified Hel1 antisera that recognizes the N terminus of the hChlR1 protein (200 ng of hChlR1 and KRm). B, cosedimentation of hChlR1 protein, DNA helicase, and DNA-dependent ATPase activities. Top,50 μg of wild type hChlR1 protein was loaded onto a 5-ml 15–40% glycerol gradient; after centrifugation for 20 h at 250,000 × g, aliquots (2 μl) of the eluted fractions were assayed for helicase activity (middle) using the 18mer-M13 helicase substrate; DNA-dependent ATPase activity (bottom). BSA, bovine serum albumin.