Mutations in the breast cancer susceptibility gene, BRCA2 , greatly increase an individual’s lifetime risk of developing breast and ovarian cancers. BRCA2 suppresses tumor formation by potentiating DNA repair via homologous recombination. Central to recombination is the assembly of a RAD51 nucleoprotein filament, which forms on single-stranded DNA (ssDNA) generated at or near the site of chromosomal damage. However, replication protein-A (RPA) rapidly binds to and continuously sequesters this ssDNA, imposing a kinetic barrier to RAD51 filament assembly that suppresses unregulated recombination. Recombination mediator proteins—of which BRCA2 is the defining member in humans—alleviate this kinetic barrier to catalyze RAD51 filament formation. We combined microfluidics, microscopy, and micromanipulation to directly measure both the binding of full-length BRCA2 to—and the assembly of RAD51 filaments on—a region of RPA-coated ssDNA within individual DNA molecules designed to mimic a resected DNA lesion common in replication-coupled recombinational repair. We demonstrate that a dimer of RAD51 is minimally required for spontaneous nucleation; however, growth self-terminates below the diffraction limit. BRCA2 accelerates nucleation of RAD51 to a rate that approaches the rapid association of RAD51 to naked ssDNA, thereby overcoming the kinetic block imposed by RPA. Furthermore, BRCA2 eliminates the need for the rate-limiting nucleation of RAD51 by chaperoning a short preassembled RAD51 filament onto the ssDNA complexed with RPA. Therefore, BRCA2 regulates recombination by initiating RAD51 filament formation.

Single-molecule biophysics and enzymology has become a vast field of investigation of biomolecules in the last decade. Application of these techniques enabled the characterization of molecular phenomena in a previously unprecedented resolution and highlighted previously hidden mechanisms of actions of enzymes. Lambda phage DNA is widely used for studying DNA biophysics and protein-DNA interactions because of its optimal size for single-molecule manipulations. However, single-molecule analysis of reactions that require specific DNA sequences or specialized DNA structures, including positioned nucleosomes to study chromatin remodeling, have been an unresolved problem. Here we demonstrate the development of a versatile lambda phage system (λKytos) modified to receive any types of DNA in the middle of lambda DNA. We integrated an array of nucleosome positioning sequences into λKytos and reconstituted fluorescently labeled nucleosomes on this substrate. Our results show that there is a strong bias for the nucleosomes to bind to the positioning sequences along the lambda DNA (30% of the nucleosomes bound to the positioning region which is 3% of the total length). The reconstituted chromatin can be visualized by optical trapping and TIRF microscopy that enables us to study sequence specific chromatin remodeling with a previously inaccessible resolution. We also demonstrate applications of the λKytos system where we introduced a replication origin, a single-stranded (ss) DNA gap, and a DNA bubble consisting of two non-pairing ssDNA region in the middle of λKytos. These substrates - and similar derivatives - will greatly facilitate single-molecule studies of DNA replication, recombination and repair in the presence and absence of reconstituted chromatin.

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. The reservoir provides the means to examine the formation of protein-DNA complexes in solution in the absence of external flow forces while maintaining a predetermined end-to-end extension of the DNA. These features facilitate the examination of the role of 3D DNA conformation and dynamics in protein-DNA interactions. Preparation of flow cells and reagents requires 2 days each; in situ DNA dumbbell assembly and imaging of single protein-DNA complexes require another day.

The Saccharomyces cerevisiae Dmc1 and Tid1 proteins are required for the pairing of homologous chromosomes during meiotic recombination. This pairing is the precursor to the formation of crossovers between homologs, an event that is necessary for the accurate segregation of chromosomes. Failure to form crossovers can have serious consequences and may lead to chromosomal imbalance. Dmc1, a meiosis-specific paralog of Rad51, mediates the pairing of homologous chromosomes. Tid1, a Rad54 paralog, although not meiosis-specific, interacts with Dmc1 and promotes crossover formation between homologs. In this study, we show that purified Dmc1 and Tid1 interact physically and functionally. Dmc1 forms stable nucleoprotein filaments that can mediate DNA strand invasion. Tid1 stimulates Dmc1-mediated formation of joint molecules. Under conditions optimal for Dmc1 reactions, Rad51 is specifically stimulated by Rad54, establishing that Dmc1-Tid1 and Rad51-Rad54 function as specific pairs. Physical interaction studies show that specificity in function is not dictated by direct interactions between the proteins. Our data are consistent with the hypothesis that Rad51-Rad54 function together to promote intersister DNA strand exchange, whereas Dmc1-Tid1 tilt the bias toward interhomolog DNA strand exchange.

Background: DNA strand exchange proteins Dmc1 and Rad51 and translocases Tid1 and Rad54 function in DNA break repair during meiosis. Results: We biochemically demonstrate that Dmc1 and Rad51 are specifically stimulated by Tid1 and Rad54, respectively. Conclusion: Dmc1-Tid1 and Rad51-Rad54 represent functional pairs for DNA pairing and joint molecule formation. Significance: The separate and independent functioning of these proteins offers insight into DNA pairing in meiosis. The Saccharomyces cerevisiae Dmc1 and Tid1 proteins are required for the pairing of homologous chromosomes during meiotic recombination. This pairing is the precursor to the formation of crossovers between homologs, an event that is necessary for the accurate segregation of chromosomes. Failure to form crossovers can have serious consequences and may lead to chromosomal imbalance. Dmc1, a meiosis-specific paralog of Rad51, mediates the pairing of homologous chromosomes. Tid1, a Rad54 paralog, although not meiosis-specific, interacts with Dmc1 and promotes crossover formation between homologs. In this study, we show that purified Dmc1 and Tid1 interact physically and functionally. Dmc1 forms stable nucleoprotein filaments that can mediate DNA strand invasion. Tid1 stimulates Dmc1-mediated formation of joint molecules. Under conditions optimal for Dmc1 reactions, Rad51 is specifically stimulated by Rad54, establishing that Dmc1-Tid1 and Rad51Rad54 function as specific pairs. Physical interaction studies show that specificity in function is not dictated by direct interactions between the proteins. Our data are consistent with the hypothesis that Rad51-Rad54 function together to promote intersister DNA strand exchange, whereas Dmc1-Tid1 tilt the bias toward interhomolog DNA strand exchange.

Genetic evidence indicates that Saccharomyces cerevisiae Sgs1, Top3, and Rmi1 resolve topologically linked intermediates arising from DNA replication and recombination. Using purified proteins, we show that Sgs1, Top3, Rmi1, and replication protein A (RPA) coordinate catenation and decatenation of dsDNA through sequential passage of single strands of DNA, establishing a unique pathway for dsDNA decatenation in eukaryotic cells. Sgs1 is required for dsDNA unwinding and, unexpectedly, also has a structural role in DNA strand passage. RPA promotes DNA unwinding by Sgs1 by trapping ssDNA, and it stimulates DNA strand passage by Top3. Paradoxically, Rmi1 has a unique regulatory capacity that slows DNA relaxation by Top3 but stimulates DNA decatenation. We establish that Rmi1 stabilizes the "open" Top3-DNA covalent complex formed as a transient intermediate of strand passage. This concerted activity of the Sgs1-Top3-Rmi1-RPA represents an important mechanism for disentangling structures resulting from the topological features of duplex DNA.

Single-molecule analysis of RecA filament assembly on its in vivo substrate, SSB-coated single-stranded DNA, reveals that a dimer of RecA is required for nucleation, and is followed by bidirectional growth of the filament through monomer addition; the recombination mediator RecOR accelerates nucleation and growth, and the addition of RecF further stimulates nucleation. An early step in the repair of DNA lesions or stalled replication forks by homologous recombination involves the pairing of homologous DNAs by the strand-exchange protein RecA. RecA from the bacterium Escherichia coli nucleates binding on single-stranded DNA (ssDNA) as a small cluster of subunits, after which it extends to form a protein–DNA filament. Until now, it has not been possible to visualize its assembly on ssDNA because of the difficulty of creating ssDNA that can be readily used in single-molecule experiments. But now Stephen Kowalczykowski and colleagues have characterized the nucleation and extension of this filament by single-molecule microscopy. The data indicate why 'mediator' proteins might be necessary to facilitate loading and growth, predictions that are confirmed by adding the bacterial RecFOR complex to the reaction. Escherichia coli RecA is the defining member of a ubiquitous class of DNA strand-exchange proteins that are essential for homologous recombination, a pathway that maintains genomic integrity by repairing broken DNA1. To function, filaments of RecA must nucleate and grow on single-stranded DNA (ssDNA) in direct competition with ssDNA-binding protein (SSB), which rapidly binds and continuously sequesters ssDNA, kinetically blocking RecA assembly2,3. This dynamic self-assembly on a DNA lattice, in competition with another protein, is unique for the RecA family compared to other filament-forming proteins such as actin and tubulin. The complexity of this process has hindered our understanding of RecA filament assembly because ensemble measurements cannot reliably distinguish between the nucleation and growth phases, despite extensive and diverse attempts2,3,4,5. Previous single-molecule assays have measured the nucleation and growth of RecA—and its eukaryotic homologue RAD51—on naked double-stranded DNA and ssDNA6,7,8,9,10,11,12; however, the template for RecA self-assembly in vivo is SSB-coated ssDNA3. Using single-molecule microscopy, here we directly visualize RecA filament assembly on single molecules of SSB-coated ssDNA, simultaneously measuring nucleation and growth. We establish that a dimer of RecA is required for nucleation, followed by growth of the filament through monomer addition, consistent with the finding that nucleation, but not growth, is modulated by nucleotide and magnesium ion cofactors. Filament growth is bidirectional, albeit faster in the 5′→3′ direction. Both nucleation and growth are repressed at physiological conditions, highlighting the essential role of recombination mediators in potentiating assembly in vivo. We define a two-step kinetic mechanism in which RecA nucleates on transiently exposed ssDNA during SSB sliding and/or partial dissociation (DNA unwrapping) and then the RecA filament grows. We further demonstrate that the recombination mediator protein pair, RecOR (RecO and RecR), accelerates both RecA nucleation and filament growth, and that the introduction of RecF further stimulates RecA nucleation.

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. Visualization is achieved by fluorescently labeling either the DNA or the protein of interest, followed by direct imaging using high-sensitivity fluorescence microscopy. We describe the sample preparation and instrumentation used to visualize the interaction of individual proteins with single molecules of DNA. As examples, we describe the application of these methods to the study of proteins involved in recombination-mediated DNA repair, a process essential for the maintenance of genomic integrity.

The mechanisms that determine bacterial shape are in many ways poorly understood. A prime example is the Lyme disease spirochete, Borrelia burgdorferi (B. burgdorferi), which mechanically couples its motility organelles, helical flagella, to its rod-shaped cell body, producing a striking flat-wave morphology. A mathematical model is developed here that accounts for the elastic coupling of the flagella to the cell cylinder and shows that the flat-wave morphology is in fact a natural consequence of the geometrical and material properties of the components. Observations of purified periplasmic flagella show two flagellar conformations. The mathematical model suggests that the larger waveform flagellum is the more relevant for determining the shape of B. burgdorferi. Optical trapping experiments were used to measure directly the mechanical properties of these spirochetes. These results imply relative stiffnesses of the two components, which confirm the predictions of the model and show that the morphology of B. burgdorferi is completely determined by the elastic properties of the flagella and cell body. This approach is applicable to a variety of other structures in which the shape of the composite system is markedly different from that of the individual components, such as coiled-coil domains in proteins and the eukaryotic axoneme.

Nearly close-packed populations of the swimming bacterium Bacillus subtilis form a collective phase, the "Zooming BioNematic" (ZBN). This state exhibits large-scale orientational coherence, analogous to the molecular alignment of nematic liquid crystals, coupled with remarkable spatial and temporal correlations of velocity and vorticity, as measured by both novel and standard applications of particle imaging velocimetry. The appearance of turbulent dynamics in a system which is nominally in the regime of Stokes flow can be understood by accounting for the local energy input by the swimmers, with a new dimensionless ratio analogous to the Reynolds number. The interaction between organisms and boundaries, and with one another, is modeled by application of the methods of regularized Stokeslets.

Recent experiments have shown large-scale dynamic coherence in suspensions of the bacterium B. subtilis, characterized by quorum polarity, collective parallel swimming of cells. To probe mechanisms leading to this, we study the response of individual cells to steric stress, and find that they can reverse swimming direction at spatial constrictions without turning the cell body. The consequences of this propensity to flip the flagella are quantified by measurements of the inward and outward swimming velocities, whose asymptotic values far from the constriction show near perfect symmetry, implying that "forwards" and "backwards" are dynamically indistinguishable, as with E. coli.