FANCJ-A349P translocates on single-stranded DNA. The Keff-ssDNA values (A) and kcat values (B) for ATP hydrolysis catalyzed by FANCJ-WT and FANCJ-A349P proteins were determined as a function of oligonucleotide dT length, as described in supplemental Methods. Error bars represent the SD of the fit to the Michaelis-Menton equation. (C) Cartoon depicting fluorescence quenching of fluorescein covalently attached to 3′ end of ssDNA molecule by ATP-driven translocation of FANCJ to a position near the fluorophore. Binding site size or the assembly state of FANCJ is not known. For simplicity, cartoon depicts initial ATP-dependent translocation of FANCJ followed by second translocation step placing FANCJ in the vicinity of fluorescein where it quenches the fluorophore; however, physical or kinetic step size is not known. (D) Fluorescence quenching experiments, performed as described in “Fluorescence quench translocation assays,” with use of FANCJ-WT, FANCJ-A349P, and FANCJ-K52R proteins.
