Figure 1.
Purification of recombinant REL1 and initial characterization of the FRET-based assay. (A) SDS-PAGE showing the purified REL151–469-KREPA251–176 complex. REL151–469 has a size of 46.9 kDa, while 6xHis-KREPA251–176 has a size of 16.5 kDa. (B) Schematic representation of the assay principle. A nicked RNA duplex consisting of guide RNA (gRNA), 5′-RNA with donor fluorophore and 3′-RNA with acceptor fluorophore serves as substrate. After incubation with REL1, ligated RNA is detected by denaturation-resistant FRET between the two fluorophores. (C) The FRET signal is dependent on ATP and REL1. The assay was carried out as described in ‘Materials and Methods’ section under ‘Initial RNA annealing and ligation conditions’, except 100 ng REL1 and the concentrations of ATP indicated were used. Means and standard deviations for three independent experiments are shown. (D) The FRET signal is dependent on gRNA. The assay was carried out as described in ‘Materials and Methods’ section under ‘Initial RNA annealing and ligation conditions’, except 200 ng REL1 were used, gRNA was added or omitted as indicated and samples were or were not denatured at 96°C before reading, as indicated. Means and standard deviations for five (+gRNA) or three (−gRNA) replicates are shown.