Single-molecule sorting of DNA helicases

Abstract

DNA helicases participate in virtually all aspects of cellular DNA metabolism by using ATP-fueled directional translocation along the DNA molecule to unwind DNA duplexes, dismantle nucleoprotein complexes, and remove non-canonical DNA structures. Post-translational modifications and helicase interacting partners are often viewed as determining factors in controlling the switch between bona fide helicase activity and other functions of the enzyme that do not involve duplex separation. The bottleneck in developing a mechanistic understanding of human helicases and their control by post-translational modifications is obtaining sufficient quantities of the modified helicase for traditional structure-functional analyses and biochemical reconstitutions. This limitation can be overcome by single-molecule analysis, where several hundred surface-tethered molecules are sufficient to obtain a complete kinetic and thermodynamic description of the helicase-mediated substrate binding and rearrangement. Synthetic oligonucleotides site-specifically labeled with Cy3 and Cy5 fluorophores can be used to create a variety of DNA substrates that can be used to characterize DNA binding, as well as helicase translocation and duplex unwinding activities. This chapter describes “single-molecule sorting”, a robust experimental approach to simultaneously quantify, and distinguish the activities of helicases carrying their native post-translational modifications. Using this technique, a DNA helicase of interest can be produced and biotinylated in human cells to enable surface-tethering for the single-molecule studies by total internal reflection fluorescence microscopy. The pool of helicases extracted from the cells is expected to contain a mixture of post-translationally modified and unmodified enzymes, and the contributions from either population can be monitored separately, but in the same experiment providing a direct route to evaluating the effect of a given modification.

Observation and Analysis of RAD51 Nucleation Dynamics at Single-Monomer Resolution

“…Our TIRF-FRET setup uses Cy3 as a donor fluorophore (excited by a 532 nm, DPSS Laser, Coherent Inc.) and Cy5 as an acceptor fluorophore. An evanescent wave generated by total internal reflection of the laser source, specifically illuminates molecules that are tethered to the surface of the observation flowcell (<100nm) (Axelrod, 2008, Bain et al, 2016). The scattered light is removed using a Cy3/Cy5 dual band-pass filter (Semrock, FF01-577/690) in the emission optical path.…”

“…Images are chromatically separated into Cy3 image (using a Chroma ET605/70m filter) and Cy5 image (using a Chroma ET700/75m filter) using a 630-nm dichroic mirror inside the dual-view system (DV2; Photometrics) (Figure 2A). An evanescent wave generated by total internal reflection of the laser source, specifically illuminates molecules that are tethered to the surface of the slide (<100nm) (Axelrod, 2008, Bain et al, 2016) (Figure 2B). …”

“…Before use, the tubes are equilibrated to room temperature for about 20 minutes in a dark chamber to prevent condensation onto the treated surface. The passivated and functionalized quartz slides and borosilicate glass coverslips are then assembled in the observation flowcells using double-sided tape and epoxy (detailed procedures for cleaning, passivating, functionalizing, and assembling the flowcells can be found in (Joo and Ha, 2012a, Bain et al, 2016)) and depicted later in (Figure 4). This procedure reduces the non-specific binding of proteins and nanoscopic impurities in buffers that might auto-fluoresce.…”

Quantifying the Assembly of Multicomponent Molecular Machines by Single-Molecule Total Internal Reflection Fluorescence Microscopy

“…The construction of the system is described in detail in (Ghoneim and Spies, 2014, Bain et al, 2016). Briefly, this system was built on an Olympus IX-71 frame (Olympus America Inc).…”

“…For bacterially expressed proteins, BAP and the presence of 100 μM biotin in the growth media is sufficient for the efficient biotinylation of the target proteins. For proteins expressed in human, baculovirus or yeast cells, the expression system must also contain a vector for the expression of BirA ligase (see (Bain et al, 2016) for a protocol for the expression of biotinylated proteins in human cells, (Honda et al, 2014) for the baculovirus expression, and (Boehm et al, 2016) for the yeast expression).…”