BRCA2 chaperones RAD51 to single molecules of RPA-coated ssDNA.

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.

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. 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.

Direct imaging of RecA nucleation and growth on single molecules of SSB-coated ssDNA.

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 DNA. 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 assembly. 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 attempts. Previous single-molecule assays have measured the nucleation and growth of RecA--and its eukaryotic homologue RAD51--on naked double-stranded DNA and ssDNA; however, the template for RecA self-assembly in vivo is SSB-coated ssDNA. 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.

Decatenation of DNA by the S. cerevisiae Sgs1-Top3-Rmi1 and RPA complex: a mechanism for disentangling chromosomes.

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.

Saccharomyces cerevisiae Dmc1 and Rad51 proteins preferentially function with Tid1 and Rad54 proteins, respectively, to promote DNA strand invasion during genetic recombination.

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.

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. 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.