Ineke Brouwer

Sliding sleeves of XRCC4–XLF bridge DNA and connect fragments of broken DNA

Non-homologous end joining (NHEJ) is the primary pathway for repairing DNA double-strand breaks (DSBs) in mammalian cells. Such breaks are formed, for example, during gene-segment rearrangements in the adaptive immune system or by cancer therapeutic agents. Although the core components of the NHEJ machinery are known, it has remained difficult to assess the specific roles of these components and the dynamics of bringing and holding the fragments of broken DNA together. The structurally similar XRCC4 and XLF proteins are proposed to assemble as highly dynamic filaments at (or near) DSBs. Here we show, using dual- and quadruple-trap optical tweezers combined with fluorescence microscopy, how human XRCC4, XLF and XRCC4–XLF complexes interact with DNA in real time. We find that XLF stimulates the binding of XRCC4 to DNA, forming heteromeric complexes that diffuse swiftly along the DNA. Moreover, we find that XRCC4–XLF complexes robustly bridge two independent DNA molecules and that these bridges are able to slide along the DNA. These observations suggest that XRCC4–XLF complexes form mobile sleeve-like structures around DNA that can reconnect the broken ends very rapidly and hold them together. Understanding the dynamics and regulation of this mechanism will lead to clarification of how NHEJ proteins are involved in generating chromosomal translocations.

Visualization and quantification of nascent RAD51 filament formation at single-monomer resolution

Significance The mechanism of RAD51-recombinase filament formation is visualized and quantified with single-molecule resolution using a combination of dual optical tweezers, fluorescence microscopy, and microfluidics. With this method, short-lived transient intermediates formed during nascent RAD51 filament assembly were observed directly. It is observed that RAD51 nuclei consisting of a variable number of monomers bind from solution to DNA, with an interaction time that increases with nucleus size. Nuclei that remain bound to DNA long enough can grow by the incorporation of additional RAD51 monomers, stabilizing the RAD51 filament. During recombinational repair of double-stranded DNA breaks, RAD51 recombinase assembles as a nucleoprotein filament around single-stranded DNA to form a catalytically proficient structure able to promote homology recognition and strand exchange. Mediators and accessory factors guide the action and control the dynamics of RAD51 filaments. Elucidation of these control mechanisms necessitates development of approaches to quantitatively probe transient aspects of RAD51 filament dynamics. Here, we combine fluorescence microscopy, optical tweezers, and microfluidics to visualize the assembly of RAD51 filaments on bare single-stranded DNA and quantify the process with single-monomer sensitivity. We show that filaments are seeded from RAD51 nuclei that are heterogeneous in size. This heterogeneity appears to arise from the energetic balance between RAD51 self-assembly in solution and the size-dependent interaction time of the nuclei with DNA. We show that nucleation intrinsically is substrate selective, strongly favoring filament formation on bare single-stranded DNA. Furthermore, we devised a single-molecule fluorescence recovery after photobleaching assay to independently observe filament nucleation and growth, permitting direct measurement of their contributions to filament formation. Our findings yield a comprehensive, quantitative understanding of RAD51 filament formation on bare single-stranded DNA that will serve as a basis to elucidate how mediators help RAD51 filament assembly and accessory factors control filament dynamics.

Direct quantitative detection of Doc2b-induced hemifusion in optically trapped membranes.

Ca2+-sensor proteins control the secretion of many neuroendocrine substances. Calcium-secretion coupling may involve several mechanisms. First, Ca2+-dependent association of their tandem C2 domains with phosphatidylserine may induce membrane curvature and thereby enhance fusion. Second, their association with SNARE complexes may inhibit membrane fusion in the absence of a Ca2+ trigger. Here we present a method using two optically trapped beads coated with SNARE-free synthetic membranes to elucidate the direct role of the C2AB domain of the soluble Ca2+-sensor Doc2b. Contacting membranes are often coupled by a Doc2b-coated membrane stalk that resists forces up to 600 pN upon bead separation. Stalk formation depends strictly on Ca2+ and phosphatidylserine. Real-time fluorescence imaging shows phospholipid but not content mixing, indicating membrane hemifusion. Thus, Doc2b acts directly on membranes and stabilizes the hemifusion intermediate in this cell-free system. In living cells, this mechanism may co-occur with progressive SNARE complex assembly, together defining Ca2+-secretion coupling.

Human RAD52 Captures and Holds DNA Strands, Increases DNA Flexibility, and Prevents Melting of Duplex DNA: Implications for DNA Recombination

Summary Human RAD52 promotes annealing of complementary single-stranded DNA (ssDNA). In-depth knowledge of RAD52-DNA interaction is required to understand how its activity is integrated in DNA repair processes. Here, we visualize individual fluorescent RAD52 complexes interacting with single DNA molecules. The interaction with ssDNA is rapid, static, and tight, where ssDNA appears to wrap around RAD52 complexes that promote intra-molecular bridging. With double-stranded DNA (dsDNA), interaction is slower, weaker, and often diffusive. Interestingly, force spectroscopy experiments show that RAD52 alters the mechanics dsDNA by enhancing DNA flexibility and increasing DNA contour length, suggesting intercalation. RAD52 binding changes the nature of the overstretching transition of dsDNA and prevents DNA melting, which is advantageous for strand clamping during or after annealing. DNA-bound RAD52 is efficient at capturing ssDNA in trans. Together, these effects may help key steps in DNA repair, such as second-end capture during homologous recombination or strand annealing during RAD51-independent recombination reactions.

Versatile Quadruple-Trap Optical Tweezers for Dual DNA Experiments

Optical manipulation techniques provide researchers the powerful ability to directly move, probe and interrogate molecular complexes. Quadruple optical trapping is an emerging method for optical manipulation and force spectroscopy that has found its primary use in studying dual DNA interactions, but is certainly not limited to DNA investigations. The key benefit of quadruple optical trapping is that two molecular strands can be manipulated independently and simultaneously. The molecular geometries of the strands can thus be controlled and their interactions can be quantified by force measurements. Accurate control of molecular geometry is of critical importance for the analysis of, for example, protein-mediated DNA-bridging, which plays an important role in DNA compaction. Here, we describe the design of a dedicated and robust quadruple optical trapping-instrument. This instrument can be switched straightforwardly to a high-resolution dual trap and it is integrated with microfluidics and single-molecule fluorescence microscopy, making it a highly versatile tool for correlative single-molecule analysis of a wide range of biomolecular systems.

Two distinct conformational states define the interaction of human RAD51‐ATP with single‐stranded DNA

An essential mechanism for repairing DNA double‐strand breaks is homologous recombination (HR). One of its core catalysts is human RAD51 (hRAD51), which assembles as a helical nucleoprotein filament on single‐stranded DNA, promoting DNA‐strand exchange. Here, we study the interaction of hRAD51 with single‐stranded DNA using a single‐molecule approach. We show that ATP‐bound hRAD51 filaments can exist in two different states with different contour lengths and with a free‐energy difference of ~4 kBT per hRAD51 monomer. Upon ATP hydrolysis, the filaments convert into a disassembly‐competent ADP‐bound configuration. In agreement with the single‐molecule analysis, we demonstrate the presence of two distinct protomer interfaces in the crystal structure of a hRAD51‐ATP filament, providing a structural basis for the two conformational states of the filament. Together, our findings provide evidence that hRAD51‐ATP filaments can exist in two interconvertible conformational states, which might be functionally relevant for DNA homology recognition and strand exchange.

Probing DNA-DNA Interactions with a Combination of Quadruple-Trap Optical Tweezers and Microfluidics

DNA metabolism and DNA compaction in vivo involve frequent interactions of remote DNA segments, mediated by proteins. In order to gain insight into such interactions, quadruple-trap optical tweezers have been developed. This technique provides an unprecedented degree of control through the ability to independently manipulate two DNA molecules in three dimensions. In this way, discrete regions of different DNA molecules can be brought into contact with one another, with a well-defined spatial configuration. At the same time, the tension and extension of the DNA molecules can be monitored. Furthermore, combining quadruple-trap optical tweezers with microfluidics makes fast buffer exchange possible, which is important for in situ generation of the dual DNA-protein constructs needed for these kinds of experiments. In this way, processes such as protein-mediated inter-DNA bridging can be studied with unprecedented control. This chapter provides a step-by-step description of how to perform a dual DNA manipulation experiment using combined quadruple-trap optical tweezers and microfluidics.

Direct Visualization and Quantification of Doc2B-Mediated Membrane Fusion

The C2 domains of the Doc2b protein are known to play a crucial role in regulating exocytosis through the coupling of calcium signals to secretory events. The exact mechanism, however, remains debated: Doc2b either inhibits SNARE-mediated membrane fusion at low calcium concentrations or it directly enhances membrane remodelling events at elevated calcium concentrations.Using a novel combination of optical trapping and fluorescence microscopy, we have addressed this issue by monitoring membrane-membrane interactions between two optically trapped micrometer-sized lipid-coated polystyrene beads. We initially brought the beads in close proximity to allow any protein-lipid and lipid-lipid interactions to take place and then retracted one bead while continuously measuring the force of the interaction between the two beads. The lipid coating on the beads was fluorescently labelled, enabling simultaneous imaging of lipid remodelling and measurement of forces associated with this process.The results show that in the presence of Doc2b, phosphatidylserine and calcium, a micrometers-long membrane stalk forms between the two individual membranes which is stable on a timescale of multiple minutes. Lipid mixing was observed, thus providing unambiguous evidence for an active role of Doc2b in membrane remodelling. The interaction forces between the two lipid-coated beads displayed a broad range up to 150 pN when a membrane stalk was formed.We conclude that Doc2b contributes to SNARE-dependent exocytosis by enhancing the probability of membrane stalk formation during local calcium elevations. In the near future, we aim to use a fluorescently labelled variant of Doc2b to determine the number of proteins bound needed to mediate the interaction and the position of the Doc2b protein with respect to the membrane stalk.