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

Abstract

DNA ends generated by double-strand breaks are vulnerable intermediates that must be rapidly recognized, protected and resolved to preserve genome integrity. We present 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 optical tweezers coupled to confocal fluorescence microscopy. Eliminating DNA surface anchoring, facilitating rapid protein and buffer exchange, and offering 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. OTCurtains 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.