Next-Generation DNA Curtains for Single-Molecule Studies of Homologous Recombination

Abstract

Homologous recombination (HR) is a universally conserved DNA double-strand break repair pathway. Single-molecule fluorescence imaging approaches have revealed new mechanistic insights into nearly all aspects of HR. These methods are especially suited for studying protein complexes because multicolor fluorescent imaging can parse out subassemblies and transient intermediates that associate with the DNA substrates on the millisecond to hour timescales. However, acquiring single-molecule datasets remains challenging because most of these approaches are designed to measure one molecular reaction at a time. The DNA curtains platform facilitates high-throughput single-molecule imaging by organizing arrays of DNA molecules on the surface of a microfluidic flowcell. Here, we describe a second-generation UV lithography-based protocol for fabricating flowcells for DNA curtains. This protocol greatly reduces the challenges associated with assembling DNA curtains and paves the way for the rapid acquisition of large datasets from individual single-molecule experiments. Drawing on our recent studies of human HR, we also provide an overview of how DNA curtains can be used for observing facilitated protein diffusion, processive enzyme translocation, and nucleoprotein filament dynamics on single-stranded DNA. Together, these protocols and case studies form a comprehensive introduction for other researchers that may want to adapt DNA curtains for high-throughput single-molecule studies of DNA replication, transcription, and repair.

Phage Mu Gam protein promotes NHEJ in concert with Escherichia coli ligase

“…The ladder of bands further indicated that multiple Gam dimers can load on the substrate, as has been observed for the eukaryotic Ku complex. Next, we employed high-throughput single-molecule DNA curtains to image MuGam as it interacts with the DNA substrate (30,31). In this assay, thousands of individual DNA molecules (∼48.5 kb long, derived from λ-phage DNA) are organized at microfabricated chromium barriers and visualized by total internal reflection fluorescence microscopy.…”

“…DNA curtains were assembled on microfabricated microscope slides, as described previously (30,31). All single-molecule data were collected at 37°C in imaging buffer [40 mM Tris·HCl (pH 8.0), 2 mM MgCl2, 1 mM ATP, 1 mM DTT, 0.2 mg·mL −1 BSA] on a Nikon Ti-E microscope in a prism total internal reflection fluorescence configuration.…”

RPA phosphorylation regulates DNA resection

“…We established a single-molecule DNA curtains assay to image the role of RPA during DNA resection (Figure 1). In this assay, BLM helicase and the nucleases EXO1 or DNA2 are imaged on 48.5 kb-long DNA molecules that are organized on the surface of a lipid-coated microfluidic flowcell (Gallardo et al, 2015;Soniat et al, 2017). One end of the DNA substrate is biotinylated and tethered to streptavidin deposited on the lipid bilayer.…”

Intrinsically disordered regions regulate both catalytic and non-catalytic activities of the MutLα mismatch repair complex

“…We examined Mlh1-Pms1 diffusion on doubletethered DNA curtains (Figure 2A-B). In this assay, a 48.5 kb-long DNA substrate is extended over a fluid lipid bilayer between two microfabricated chromium barriers (51,60,61). The lipid bilayer provides a biomimetic surface that passivates the flowcell surface from non-specific adsorption by DNA-binding proteins.…”