Single-Stranded DNA Curtains for Studying the Srs2 Helicase Using Total Internal Reflection Fluorescence Microscopy

Abstract

Helicases are crucial participants in many types of DNA repair reactions, including homologous recombination. The properties of these enzymes can be assayed by traditional bulk biochemical analysis; however, these types of assays cannot directly access some types of information. In particular, bulk biochemical assays cannot readily access information that may be obscured in population averages. Single-molecule assays offer the potential advantage of being able to visualize the molecules in question in real time, thus providing direct access to questions relating to translocation velocity, processivity, and insights into how helicases may behave on different types of substrates. Here, we describe the use of single-stranded DNA (ssDNA) curtains as an assay for directly viewing the behavior of the Saccharomyces cerevisiae Srs2 helicase on single molecules of ssDNA. When used with total internal reflection fluorescence microscopy, these methods can be used to track the binding and movements of individual helicase complexes, and allow new insights into helicase behaviors at the single-molecule level.

Meiosis-specific recombinase Dmc1 is a potent inhibitor of the Srs2 antirecombinase

“…We have established ssDNA curtain assays for visualizing the behaviors of Srs2 on HR intermediates in real time (Fig. 1C) (42,44,45). In brief, long ssDNA substrates (≥50 kilonucleotides) are generated by rolling circle replication using a 5′ biotinylated primer (45)(46)(47).…”

“…1C) (42,44,45). In brief, long ssDNA substrates (≥50 kilonucleotides) are generated by rolling circle replication using a 5′ biotinylated primer (45)(46)(47). The resulting 5′ biotinylated ssDNA is tethered to a supported lipid bilayer on the surface of a microfluidic sample chamber through a biotin-streptavidin linkage (45)(46)(47).…”

“…In brief, long ssDNA substrates (≥50 kilonucleotides) are generated by rolling circle replication using a 5′ biotinylated primer (45)(46)(47). The resulting 5′ biotinylated ssDNA is tethered to a supported lipid bilayer on the surface of a microfluidic sample chamber through a biotin-streptavidin linkage (45)(46)(47). The 5′ ends of the ssDNA are then aligned at nanofabricated Cr barriers to lipid diffusion, and the downstream ends of the ssDNA are attached to Cr pedestals, which are deposited onto the fused silica by electronbeam lithography (45)(46)(47).…”

“…The resulting 5′ biotinylated ssDNA is tethered to a supported lipid bilayer on the surface of a microfluidic sample chamber through a biotin-streptavidin linkage (45)(46)(47). The 5′ ends of the ssDNA are then aligned at nanofabricated Cr barriers to lipid diffusion, and the downstream ends of the ssDNA are attached to Cr pedestals, which are deposited onto the fused silica by electronbeam lithography (45)(46)(47). Addition of GFP-or mCherry-RPA allows the ssDNA to be extended by hydrodynamic force, and also provides means of visualizing the ssDNA by total internal reflection fluorescence microscopy (45)(46)(47).…”

Spontaneous self-segregation of Rad51 and Dmc1 DNA recombinases within mixed recombinase filaments

“…For all two-color images, we use a custom-built shuttering system to avoid the bleed through from the green into the red channel during image acquisition. With this system, images from the green (GFP) and the red (mCherry) channels are recorded independently, these recordings are offset by 100 milliseconds such that when one camera records the red channel image, the green laser is shuttered off, and vice versa (30)(31)(32).…”