OT-Curtains: an approach for studying protein interactions with DNA ends using optical tweezers and confocal fluorescence microscopy.

DNA ends generated by double-strand breaks are vulnerable intermediates that must be rapidly recognized, protected, and resolved to preserve genome integrity. We present optical tweezers (OT)-Curtains, a single-molecule method inspired by DNA curtains that uses a custom branched DNA substrate containing multiple accessible ends for simultaneous observation on dual-trap OT coupled to confocal fluorescence microscopy. Eliminating DNA surface anchoring, facilitating rapid protein and buffer exchange, and offering the possibility for force-free experiments, OT-Curtains overcomes common limitations of flow-stretch-based methods. OT-Curtains allows real-time visualization and quantification of end recognition, protection, resection, and cleavage at several DNA ends in parallel. We demonstrate compatibility with well-studied DNA-binding systems by monitoring Ku-mediated DNA break recognition, AddAB-mediated DNA break resection, ParB-mediated DNA condensation, and KpnI-mediated DNA cleavage. We show that kinetic and mechanistic parameters can be extracted from the data under defined forces and solution conditions. OT-Curtains offers an accessible and multiplexed route to interrogate DNA-end transactions central to double-stranded DNA break repair pathways and telomere biology, as well as a general framework for benchmarking proteins acting at DNA ends.