Single-Molecule Förster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Presented here is a protocol for performing single-molecule Förster resonance energy transfer to study HJ resolution. Two-color alternating excitation is used for determining the dissociation constants. Single-color time lapse smFRET is then applied in real-time cleavage assays to obtain the dwell time distribution prior to HJ resolution.