Figure 1. Biochemical analysis of purified recombinant human RECQ1 proteins.

(A) Schematic of human RECQ1 showing conserved domains and helicase core signature motifs. The conserved aromatic-rich sequence between motifs II and III is shown with a sequence alignment of the five human RecQ helicases. The two invariant aromatic amino acids and corresponding amino acid substitutions in RECQ1 are indicated in pink and blue, respectively (left). Coomassie-stained SDS polyacrylamide gel showing recombinant human RECQ1 proteins purified from insect cells (right). (B) Autoradiogram showing representative gel from EMSA experiments with RECQ1 proteins (0.15, 0.31, 0.62, 1.25, 2.5, 5, 10, and 20 nM monomer) incubated with a forked 19 bp duplex DNA substrate. (C) Apparent dissociation constant values (K_d) estimating binding affinities of RECQ1 proteins for the forked duplex substrate. (D) Kinetic constants for ATP hydrolysis by RECQ1 proteins. (E) Autoradiogram showing representative native polyacrylamide gel from helicase assays with RECQ1 proteins (0.15, 0.31, 0.62, 1.25, 2.5, 5, 10, 20 and 40 nM monomer) and the 19 bp forked duplex DNA substrate. (F) Autoradiogram showing representative native polyacrylamide gel from BM assays with RECQ1 proteins (25, 50,100, and 200 nM monomer) and mobile three-stranded D-loop DNA substrate. (G) Autoradiogram showing representative native polyacrylamide gel from BM assays with RECQ1 proteins (0.62, 1.25, 2.5, 5, 10, and 20 nM monomer) and the four-stranded HJ DNA substrate. (H) Autoradiogram showing representative native polyacrylamide gel from strand annealing assays with RECQ1 proteins (1.25, 2.5, 5, 10, 20, and 40 nM monomer) and complementary single-stranded oligonucleotides in the absence or presence of 1 mM ATPγS as indicated. (I, J) Size exclusion chromatogram showing distribution of purified RECQ1 proteins incubated in the absence (I) or presence (J) of 1 mM ATP.