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The repair of DNA by homologous recombination is an essential, efficient, and high-fidelity process that mends DNA lesions formed during cellular metabolism; these lesions include double-stranded DNA breaks, daughter-strand gaps, and DNA cross-links. Genetic defects in the homologous recombination pathway undermine genomic integrity and cause the accumulation of gross chromosomal abnormalities—including rearrangements, deletions, and aneuploidy—that contribute to cancer formation. Recombination proceeds through the formation of joint DNA molecules—homologously paired but metastable DNA intermediates that are processed by several alternative subpathways—making recombination a versatile and robust mechanism to repair damaged chromosomes. Modern biophysical methods make it possible to visualize, probe, and manipulate the individual molecules participating in the intermediate steps of recombination, revealing new details about the mechanics of genetic recombination. We review and discuss the individual stages of homologous recombination, focusing on common pathways in bacteria, yeast, and humans, and place particular emphasis on the molecular mechanisms illuminated by single-molecule methods.
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Supplemental Video 1: Unwinding of a single molecule of λ DNA by RecBCD.
This video shows unwinding of a single molecule of λ DNA – stained with YOYO-1, attached to a 1-μm polystyrene bead (stained nonspecifically by YOYO-1), and captured by an optical trap, by a single RecBCD enzyme at 37°C in the presence of 1 mM ATP; DNA is extend by solution flow from the bottom to the top. This molecule unwound at ∼440 bp/sec, and dissociated after ∼26,800 bp. Published with permissions from Reference 30.
Supplemental Video 2: Unwinding of a single molecule of DNA by RecBCD.
This video shows translocation of an individual RecBCD molecule along χ-containing λ DNA, visualized by virtue of a fluorescent 40-nm particle attached to a biotinylated RecD subunit. Solution flow is left to right. The very bright spot to the left is the 1-μm polystyrene bead, to which nanoparticles are bound nonspecifically, in the optical trap. Note that the nanoparticle--RecBCD complex can be seen to pause for about 6.6 s, and then it continues to translocate but at a much reduced speed (∼145 bp/sec versus ∼1,120 bp/sec prior to χ-recognition). Published with permission from Reference 54.
Supplemental Video 3: Interrupting and restarting single molecules of RecBCD during DNA unwinding.
This video compares unwinding of three individual molecules of λ DNA by three different single molecules of RecBCD, whose initial velocities are comparable. Solution flow is left to right. For each, the bright spot to the left is the 1-μm polystyrene bead, to which YOYO-1 is bound nonspecifically, in the optical trap. Unwinding is transiently paused by moving the molecules into a solution of EDTA (denoted by “Paused” during the video) and then resumed by returning the molecules to a solution of Mg2+:ATP. The change in relative unwinding rate is visually evident. Videos are representative of data collected and reported in Reference 31, although the video itself is previously unpublished.
Supplemental Video 4: Unwinding of a single molecule of λ DNA by RecQ, detected using fluorescent SSB and imaged using TIRF microscopy.
Initially, the dsDNA, tethered at each end to the surface of a flow cell, is fluorescent due to intercalation of the YO-PRO-1 dye molecules. As a solution containing 200 mM NaCl is introduced into the flow cell, the dye dissociates and fluorescence disappears. Subsequently, a solution containing 80 nM RecQ and SSBAF488 is introduced into the flow cell to initiate unwinding. The solution fills the channel 24 s into the video. The flow, when on, goes from left to right in the video. The elapsed time is indicated in hours:minutes:seconds and scale bar is 5 μm. Published with permission from Reference 73.
Supplemental Video 5: Salt-induced intramolecular condensation of SSB-ssDNA imaged using TIRF microscopy.
Video of a single molecule of SSBAF488-coated λ ssDNA, tethered at one end and imaged using TIRF microscopy, contracting in length upon increasing [NaOAc] from 0 to 100 mM. Solution flow is left to right. The video frames were rendered into a topological intensity map. Time zero corresponds to the time at which the pump was turned on. The dead time of the experiment was approximately 25 s due to the volume in the lines between the syringe valve and the microfluidic chamber. Published with permission from Reference 102.
Supplemental Video 6: Condensation of SSB in the absence of free protein during a transient increase from 0 to 100 mM NaOAc.
Video of a single molecule of SSBAF488-coated λ ssDNA contracting in length as the salt concentration is increased from 0 to 100 mM NaOAc, and then subsequently reduced back to zero mM, conducted in the absence of free SSBAF488. Solution flow is left to right. The flow cell was extensively washed with buffer to remove free SSB protein before beginning the experiment. Video recording began when the pump was turned on, requiring ∼40-50 s for the dead volume to be flushed from the lines to the flow chamber. SSBAF488 was omitted from both of the high-salt washes and from the 0 mM wash. Published with permission from Reference 102.
Supplemental Video 7: Direct imaging of nucleation and growth of RecA on SSB-coated ssDNA using TIRF microscopy.
This video first shows a flow-extended single molecule of 3′-biotinylated λ ssDNA coated with a fluorescent protein, SSBAF488. The molecule was tethered to a streptavidin-coated glass surface within a microfluidic flow cell and visualized using total internal reflection fluorescence microscopy. Solution flow is left to right. SSBAF488 was exchanged with non-fluorescent SSB on the ssDNA in situ, and filament assembly was initiated by injecting fluorescein labeled RecA (350 nM) with nucleotide cofactor (ATPγS). Filament assembly proceeded primarily in the absence of flow or laser excitation, which were both used only during brief intermittent, time-lapsed imaging. Images were processed and rendered into a topographical intensity map for clarity. Published with permission from Reference 110.
Supplemental Video 8: Optical trapping and manipulation of single molecules of gapped λ DNA for direct imaging of RecA filament assembly.
This video first shows 1-μm streptavidin-coated polystyrene beads flowing through Channel 1 of a multichannel, microfluidic flow chamber and the subsequent isolation of two beads by a split-beam dual optical trap (Step 1). Solution flow is left to right. The beads are then transferred to Channel 2, which contains gapped λ DNA molecules comprising 8,155 nucleotides of SSB-coated ssDNA flanked by 21.08 and 24.59 kbp of YOYO-1 stained dsDNA; the gapped DNA is biotinylated at each of the molecule, and is captured in situ by binding to the streptavidin-coated beads (Step 2). The molecule is then transferred to a DNA-free Channel 3 where the distal end of the flow-extended molecule is captured by the other bead which is micromanipulated using a steerable mirror in line with one of the infrared laser beams (Step 3). The molecule is then rotated perpendicular to flow and imaged in buffer optimized for visualizing YOYO-1 stained dsDNA in Channel 4 (Step 4). The molecule is then transferred to Channel 5 containing Mg2+:ATPγS, which accelerates YOYO-1 dissociation (Step 6). The molecule was then successively incubated in reaction buffer containing fluorescein labeled RecA, Mg2+:ATPγS, and either RecOR or RecFOR and imaged in Channel 5 to measure the rates of nucleation and growth (Step 7). Published with permission from Reference 110.
Supplemental Video 9: Composite video depicting the experimental procedure used to visualize DNA pairing on single λ DNA-dumbbell molecules by optical trapping.
A DNA pairing reaction (2 min) was performed with the 430 nt substrate at a 2 μm bead distance. Text and illustrations were inserted at appropriate places to facilitate description. A four-channel flow cell with a flow-free reservoir was used. Solution flow is top to bottom. First, two 1-μm streptavidin-coated polystyrene beads are captured in dual optical traps. Next, a single DNA molecule is captured on one bead. The DNA-dumbbell is made by sliding the DNA along the other bead, until the biotinylated end attaches. The DNA end-to-end distance is set, and the YOYO-1 dye is removed. The DNA-dumbbell is incubated with fluorescent RecA nucleoprotein filaments in the flow-free reservoir for 2 min. Afterward, the DNA-dumbbell is moved to the observation channel and is extended to near contour length to image the pairing products. Published with permission from Reference 152.
Supplemental Video 10: Dissociation of heterologously paired RecA nucleoprotein filaments during a DNA pairing experiment.
Video, performed as in Supplemental Video 9, showing RecA nucleoprotein filaments, both heterologously bound and homologously bound (left and right red spots, respectively) during the extension step of a pairing assay performed using the 1,762 nt homologous ssDNA. As the beads are separated, several loop-release events are observed involving the heterologously bound filament (left) before its dissociation from λ DNA, whereas the homologously bound RecA nucleoprotein filament (right) remains stably bound. Published with permission from Reference 152.