Exploring protein-DNA interactions in 3D using in situ construction, manipulation and visualization of individual DNA dumbbells with optical traps, microfluidics and fluorescence microscopy.

In this protocol, we describe a procedure to generate 'DNA dumbbells'-single molecules of DNA with a microscopic bead attached at each end-and techniques for manipulating individual DNA dumbbells. We also detail the design and fabrication of a microfluidic device (flow cell) used in conjunction with dual optical trapping to manipulate DNA dumbbells and to visualize individual protein-DNA complexes by single-molecule epifluorescence microscopy. Our design of the flow cell enables the rapid movement of trapped molecules between laminar flow channels and a flow-free reservoir.

Watching individual proteins acting on single molecules of DNA.

In traditional biochemical experiments, the behavior of individual proteins is obscured by ensemble averaging. To better understand the behavior of proteins that bind to and/or translocate on DNA, we have developed instrumentation that uses optical trapping, microfluidic solution delivery, and fluorescent microscopy to visualize either individual proteins or assemblies of proteins acting on single molecules of DNA. The general experimental design involves attaching a single DNA molecule to a polystyrene microsphere that is then used as a microscopic handle to manipulate individual DNA molecules with a laser trap.

Single molecule analysis of a red fluorescent RecA protein reveals a defect in nucleoprotein filament nucleation that relates to its reduced biological functions.

Fluorescent fusion proteins are exceedingly useful for monitoring protein localization in situ or visualizing protein behavior at the single molecule level. Unfortunately, some proteins are rendered inactive by the fusion. To circumvent this problem, we fused a hyperactive RecA protein (RecA803 protein) to monomeric red fluorescent protein (mRFP1) to produce a functional protein (RecA-RFP) that is suitable for in vivo and in vitro analysis.

The BRC repeats of BRCA2 modulate the DNA-binding selectivity of RAD51.

The breast cancer susceptibility protein, BRCA2, is essential for recombinational DNA repair. BRCA2 delivers RAD51 to double-stranded DNA (dsDNA) breaks through interaction with eight conserved, approximately 35 amino acid motifs, the BRC repeats. Here we show that the solitary BRC4 promotes assembly of RAD51 onto single-stranded DNA (ssDNA), but not dsDNA, to stimulate DNA strand exchange.

Direct imaging of human Rad51 nucleoprotein dynamics on individual DNA molecules.

Rad51 protein (Rad51) is central to recombinational repair of double-strand DNA breaks. It polymerizes onto DNA and promotes strand exchange between homologous chromosomes. We visualized the real-time assembly and disassembly of human Rad51 nucleoprotein filaments on double-stranded DNA by single-molecule fluorescence microscopy.

RecBCD enzyme switches lead motor subunits in response to chi recognition.

RecBCD is a DNA helicase comprising two motor subunits, RecB and RecD. Recognition of the recombination hotspot, chi, causes RecBCD to pause and reduce translocation speed. To understand this control of translocation, we used single-molecule visualization to compare RecBCD to the RecBCD(K177Q) mutant with a defective RecD motor.

Single molecule imaging of Tid1/Rdh54, a Rad54 homolog that translocates on duplex DNA and can disrupt joint molecules.

The Saccharomyces cerevisiae Tid1 protein is important for the recombinational repair of double-stranded DNA breaks during meiosis. Tid1 is a member of Swi2/Snf2 family of chromatin remodeling proteins and shares homology with Rad54. Members of this family hydrolyze ATP and promote 1) chromatin remodeling, 2) DNA topology alterations, and 3) displacement of proteins from DNA.

Direct observation of individual RecA filaments assembling on single DNA molecules.

Escherichia coli RecA is essential for the repair of DNA double-strand breaks by homologous recombination. Repair requires the formation of a RecA nucleoprotein filament. Previous studies have indicated a mechanism of filament assembly whereby slow nucleation of RecA protein on DNA is followed by rapid growth.

Visualization of Rad54, a chromatin remodeling protein, translocating on single DNA molecules.

Rad54 protein plays an important role in the recombinational repair of double-strand DNA (dsDNA) breaks. It is a dsDNA-dependent ATPase that belongs to the Swi2/Snf2 family of chromatin-remodeling proteins. Rad54 remodels (1) DNA structure, (2) chromatin structure, and (3) Rad51-dsDNA complexes.