Turning the Mre11/Rad50 DNA repair complex on its head: lessons from SMC protein hinges, dynamic coiled-coil movements and DNA loop-extrusion?

The bacterial SbcC/SbcD DNA repair proteins were identified over a quarter of a century ago. Following the subsequent identification of the homologous Mre11/Rad50 complex in the eukaryotes and archaea, it has become clear that this conserved chromosomal processing machinery is central to DNA repair pathways and the maintenance of genomic stability in all forms of life. A number of experimental studies have explored this intriguing genome surveillance machinery, yielding significant insights and providing conceptual advances towards our understanding of how this complex operates to mediate DNA repair. However, the inherent complexity and dynamic nature of this chromosome-manipulating machinery continue to obfuscate experimental interrogations, and details regarding the precise mechanisms that underpin the critical repair events remain unanswered. This review will summarize our current understanding of the dramatic structural changes that occur in Mre11/Rad50 complex to mediate chromosomal tethering and accomplish the associated DNA processing events. In addition, undetermined mechanistic aspects of the DNA enzymatic pathways driven by this vital yet enigmatic chromosomal surveillance and repair apparatus will be discussed. In particular, novel and putative models of DNA damage recognition will be considered and comparisons will be made between the modes of action of the Rad50 protein and other related ATPases of the overarching SMC superfamily.

Modes of action of the archaeal Mre11/Rad50 DNA-repair complex revealed by fast-scan atomic force microscopy.

Mre11 and Rad50 (M/R) proteins are part of an evolutionarily conserved macromolecular apparatus that maintains genomic integrity through repair pathways. Prior structural studies have revealed that this apparatus is extremely dynamic, displaying flexibility in the long coiled-coil regions of Rad50, a member of the structural maintenance of chromosome (SMC) superfamily of ATPases. However, many details of the mechanics of M/R chromosomal manipulation during DNA-repair events remain unclear. Here, we investigate the properties of the thermostable M/R complex from the archaeon Sulfolobus acidocaldarius using atomic force microscopy (AFM) to understand how this macromolecular machinery orchestrates DNA repair. While previous studies have observed canonical interactions between the globular domains of M/R and DNA, we observe transient interactions between DNA substrates and the Rad50 coiled coils. Fast-scan AFM videos (at 1-2 frames per second) of M/R complexes reveal that these interactions result in manipulation and translocation of the DNA substrates. Our study also shows dramatic and unprecedented ATP-dependent DNA unwinding events by the M/R complex, which extend hundreds of base pairs in length. Supported by molecular dynamic simulations, we propose a model for M/R recognition at DNA breaks in which the Rad50 coiled coils aid movement along DNA substrates until a DNA end is encountered, after which the DNA unwinding activity potentiates the downstream homologous recombination (HR)-mediated DNA repair.

DNA Nanostructures for Targeted Antimicrobial Delivery.

We report the use of DNA origami nanostructures, functionalized with aptamers, as a vehicle for delivering the antibacterial enzyme lysozyme in a specific and efficient manner. We test the system against Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) targets. We use direct stochastic optical reconstruction microscopy (dSTORM) and atomic force microscopy (AFM) to characterize the DNA origami nanostructures and structured illumination microscopy (SIM) to assess the binding of the origami to the bacteria. We show that treatment with lysozyme-functionalized origami slows bacterial growth more effectively than treatment with free lysozyme. Our study introduces DNA origami as a tool in the fight against antibiotic resistance, and our results demonstrate the specificity and efficiency of the nanostructure as a drug delivery vehicle.

Molecular architecture of the SYCP3 fibre and its interaction with DNA.

The synaptonemal complex (SC) keeps homologous chromosomes in close alignment during meiotic recombination. A hallmark of the SC is the presence of its constituent protein SYCP3 on the chromosome axis. During SC assembly, SYCP3 is deposited on both axes of the homologue pair, forming axial elements that fuse into the lateral element (LE) in the tripartite structure of the mature SC. We have used cryo-electron tomography and atomic force microscopy to study the mechanism of assembly and DNA binding of the SYCP3 fibre. We find that the three-dimensional architecture of the fibre is built on a highly irregular arrangement of SYCP3 molecules displaying very limited local geometry. Interaction between SYCP3 molecules is driven by the intrinsically disordered tails of the protein, with no contact between the helical cores, resulting in a flexible fibre assembly. We demonstrate that the SYCP3 fibre can engage in extensive interactions with DNA, indicative of an efficient mechanism for incorporation of DNA within the fibre. Our findings suggest that SYCP3 deposition on the chromosome axis might take place by polymerization into a fibre that is fastened to the chromosome surface via DNA binding.

Sar1 GTPase Activity Is Regulated by Membrane Curvature.

The majority of biosynthetic secretory proteins initiate their journey through the endomembrane system from specific subdomains of the endoplasmic reticulum. At these locations, coated transport carriers are generated, with the Sar1 GTPase playing a critical role in membrane bending, recruitment of coat components, and nascent vesicle formation. How these events are appropriately coordinated remains poorly understood. Here, we demonstrate that Sar1 acts as the curvature-sensing component of the COPII coat complex and highlight the ability of Sar1 to bind more avidly to membranes of high curvature. Additionally, using an atomic force microscopy-based approach, we further show that the intrinsic GTPase activity of Sar1 is necessary for remodeling lipid bilayers. Consistent with this idea, Sar1-mediated membrane remodeling is dramatically accelerated in the presence of its guanine nucleotide-activating protein (GAP), Sec23-Sec24, and blocked upon addition of guanosine-5'-[(ß,γ)-imido]triphosphate, a poorly hydrolysable analog of GTP. Our results also indicate that Sar1 GTPase activity is stimulated by membranes that exhibit elevated curvature, potentially enabling Sar1 membrane scission activity to be spatially restricted to highly bent membranes that are characteristic of a bud neck. Taken together, our data support a stepwise model in which the amino-terminal amphipathic helix of GTP-bound Sar1 stably penetrates the endoplasmic reticulum membrane, promoting local membrane deformation. As membrane bending increases, Sar1 membrane binding is elevated, ultimately culminating in GTP hydrolysis, which may destabilize the bilayer sufficiently to facilitate membrane fission.

Structural dynamics of single molecules studied with high-speed atomic force microscopy.

INTRODUCTION: Atomic force microscopy (AFM) is a scanning probe technique that has been in use in biology to generate sub-nanometre resolution images in near-physiological environments for over 20 years. Most AFM work uses instruments that take several minutes to generate each image but instruments that can produce real-time images have recently become available and there is now a reasonable body of work published on this technique. The importance of this high-speed AFM is that dynamic events of individual macromolecules can be studied. AREAS COVERED: This review focuses on specific examples that demonstrate the potential of the technique. It covers four areas in which high-speed AFM has been used to elucidate mechanisms that are either unstudied or not clearly understood. These areas are: protein-protein interactions; DNA-protein interactions; quantification of biological processes; the use of DNA origami scaffolds as nanostructures to build and study dynamic molecular events. EXPERT OPINION: High-speed AFM shares advantages and disadvantages with conventional AFM, but it compares well in quality of data generated and in ease of use with other currently available techniques of high-resolution biological imaging. As the instruments become more widespread, the value of high-speed AFM and its potential to complement other techniques in molecular and cell biology should become more appreciated.

Visualization of structural changes accompanying activation of N-methyl-D-aspartate (NMDA) receptors using fast-scan atomic force microscopy imaging.

NMDA receptors are widely expressed in the central nervous system and play a major role in excitatory synaptic transmission and plasticity. Here, we used atomic force microscopy (AFM) imaging to visualize activation-induced structural changes in the GluN1/GluN2A NMDA receptor reconstituted into a lipid bilayer. In the absence of agonist, AFM imaging revealed two populations of particles with heights above the bilayer surface of 8.6 and 3.4 nm. The taller, but not the shorter, particles could be specifically decorated by an anti-GluN1 antibody, which recognizes the S2 segment of the agonist-binding domain, indicating that the two populations represent the extracellular and intracellular regions of the receptor, respectively. In the presence of glycine and glutamate, there was a reduction in the height of the extracellular region to 7.3 nm. In contrast, the height of the intracellular domain was unaffected. Fast-scan AFM imaging combined with UV photolysis of caged glutamate permitted the detection of a rapid reduction in the height of individual NMDA receptors. The reduction in height did not occur in the absence of the co-agonist glycine or in the presence of the selective NMDA receptor antagonist D(-)-2-amino-5-phosphonopentanoic acid, indicating that the observed structural change was caused by receptor activation. These results represent the first demonstration of an activation-induced effect on the structure of the NMDA receptor at the single-molecule level. A change in receptor size following activation could have important functional implications, in particular by affecting interactions between the NMDA receptor and its extracellular synaptic partners.

Demonstration of ligand decoration, and ligand-induced perturbation, of G-quadruplexes in a plasmid using atomic force microscopy.

G-Quadruplexes are nucleic acid secondary structures consisting of a planar arrangement of four guanine residues. Potential G-quadruplex-forming sequences are widely distributed throughout the genome. Significantly, they are present in telomeres and are enriched in gene promoters and first introns, raising the possibility that perturbation of G-quadruplex stability might have therapeutic potential, for example in the treatment of cancer. Ligands that interact selectively with G-quadruplexes include both proteins and small molecules, although the interactions between ligands and their G-quadruplex targets have been monitored using indirect methods. In addition, the G-quadruplex targets have often been short DNA fragments. Here, we have used atomic force microscopy imaging to examine directly at the single-molecule level the interaction of ligands with G-quadruplexes generated during transcription of a plasmid containing a G-rich insert. We show that the structures produced during transcription are decorated specifically by the single-chain antibody HF1 and by the nuclear protein PARP-1, both of which are known to recognize G-quadruplexes. Our results provide clear structural evidence of G-quadruplex formation in a transcription-dependent case and demonstrate directly how small-molecule stabilizers and destabilizers can manipulate these structures in a biochemically functional system.

DNA translocation by type III restriction enzymes: a comparison of current models of their operation derived from ensemble and single-molecule measurements.

Much insight into the interactions of DNA and enzymes has been obtained using a number of single-molecule techniques. However, recent results generated using two of these techniques-atomic force microscopy (AFM) and magnetic tweezers (MT)-have produced apparently contradictory results when applied to the action of the ATP-dependent type III restriction endonucleases on DNA. The AFM images show extensive looping of the DNA brought about by the existence of multiple DNA binding sites on each enzyme and enzyme dimerisation. The MT experiments show no evidence for looping being a requirement for DNA cleavage, but instead support a diffusive sliding of the enzyme on the DNA until an enzyme-enzyme collision occurs, leading to cleavage. Not only do these two methods appear to disagree, but also the models derived from them have difficulty explaining some ensemble biochemical results on DNA cleavage. In this 'Survey and Summary', we describe several different models put forward for the action of type III restriction enzymes and their inadequacies. We also attempt to reconcile the different models and indicate areas for further experimentation to elucidate the mechanism of these enzymes.

Structure and binding mechanism of vascular endothelial cadherin: a divergent classical cadherin.

Vascular endothelial cadherin (VE-cadherin), a divergent member of the type II classical cadherin family of cell adhesion proteins, mediates homophilic adhesion in the vascular endothelium. Previous investigations with a bacterially produced protein suggested that VE-cadherin forms cell surface trimers that bind between apposed cells to form hexamers. Here we report studies of mammalian-produced VE-cadherin ectodomains suggesting that, like other classical cadherins, VE-cadherin forms adhesive trans dimers between monomers located on opposing cell surfaces. Trimerization of the bacterially produced protein appears to be an artifact that arises from a lack of glycosylation. We also present the 2.1-Å-resolution crystal structure of the VE-cadherin EC1-2 adhesive region, which reveals homodimerization via the strand-swap mechanism common to classical cadherins. In common with type II cadherins, strand-swap binding involves two tryptophan anchor residues, but the adhesive interface resembles type I cadherins in that VE-cadherin does not form a large nonswapped hydrophobic surface. Thus, VE-cadherin is an outlier among classical cadherins, with characteristics of both type I and type II subfamilies.

Visual analysis of concerted cleavage by type IIF restriction enzyme SfiI in subsecond time region.

Many DNA regulatory factors require communication between distantly separated DNA sites for their activity. The type IIF restriction enzyme SfiI is often used as a model system of site communication. Here, we used fast-scanning atomic force microscopy to monitor the DNA cleavage process with SfiI and the changes in the single SfiI-DNA complex in the presence of either Mg²âº or Ca²âº at a scan rate of 1-2 fps. The increased time resolution allowed us to visualize the concerted cleavage of the protein at two cognate sites. The four termini generated by the cleavage were released in a multistep manner. The high temporal resolution enabled us to visualize the translocation of a DNA strand on a looped complex and intersegmental transfer of the SfiI protein in which swapping of the site is performed without protein dissociation. On the basis of our results, we propose that the SfiI tetramer can remain bound to one of the sites even after cleavage, allowing the other site on the DNA molecule to fill the empty DNA-binding cleft by combining a one-dimensional diffusion-mediated sliding and a segment transfer mechanism.

Interaction of synaptotagmin with lipid bilayers, analyzed by single-molecule force spectroscopy.

Synaptotagmin I is the major Ca²(+) sensor for membrane fusion during neurotransmitter release. The cytoplasmic domain of synaptotagmin consists of two C2 domains, C2A and C2B. On binding Ca²(+), the tips of the two C2 domains rapidly and synchronously penetrate lipid bilayers. We investigated the forces of interaction between synaptotagmin and lipid bilayers using single-molecule force spectroscopy. Glutathione-S-transferase-tagged proteins were attached to an atomic force microscope cantilever via a glutathione-derivatized polyethylene glycol linker. With wild-type C2AB, the force profile for a bilayer containing phosphatidylserine had both Ca²(+)-dependent and Ca²(+)-independent components. No force was detected when the bilayer lacked phosphatidylserine, even in the presence of Ca²(+). The binding characteristics of C2A and C2B indicated that the two C2 domains cooperate in binding synaptotagmin to the bilayer, and that the relatively weak Ca²(+)-independent force depends only on C2A. When the lysine residues K189-192 and K326, 327 were mutated to alanine, the strong Ca²(+)-dependent binding interaction was either absent or greatly reduced. We conclude that synaptotagmin binds to the bilayer via C2A even in absence of Ca²(+), and also that positively charged regions of both C2A and C2B are essential for the strong Ca²(+)-dependent binding of synaptotagmin to the bilayer.

Acid-sensing ion channel (ASIC) 1a undergoes a height transition in response to acidification.

The acid-sensing ion channel (ASIC) 1a is known to assemble as a homotrimer. Here, we used atomic force microscopy to image ASIC1a, integrated into lipid bilayers, at pH 7.0 and pH 6.0. The triangular appearance of the channel was clearly visible. A height distribution for the channels at pH 7.0 had two peaks, at 2 and 4 nm, likely representing the intracellular and extracellular domains, respectively. At pH 6.0 the 2-nm peak remained, but the higher peak shifted to 6 nm. Hence, the extracellular domain of the channel becomes 'taller' after acidification.

Demonstration of a direct interaction between sigma-1 receptors and acid-sensing ion channels.

The sigma-1 receptor is a widely expressed protein that interacts with a variety of ion channels, including the acid-sensing ion channel (ASIC) 1a. Here we used atomic force microscopy to determine the architecture of the ASIC1a/sigma-1 receptor complex. When isolated His(8)-tagged ASIC1a was imaged in complex with anti-His(6) antibodies, the angle between pairs of bound antibodies was 135 degrees , consistent with the known trimeric structure of the channel. When ASIC1a was coexpressed with FLAG/His(6)-tagged sigma-1 receptor, ASIC1a became decorated with small particles, and pairs of these particles bound at an angle of 131 degrees . When these complexes were incubated with anti-FLAG antibodies, pairs of antibodies bound at an angle of 134 degrees , confirming that the small particles were sigma-1 receptors. Of interest, we found that the sigma-1 receptor ligand haloperidol caused an approximately 50% reduction in ASIC1a/sigma-receptor binding, suggesting a way in which sigma-1 ligands might modulate channel properties. For the first time, to our knowledge, we have resolved the structure of a complex between the sigma-1 receptor and a target ion channel, and demonstrated that the stoichiometry of the interaction is 1 sigma-1 receptor/1 ASIC1a subunit.

Direct visualization of G-quadruplexes in DNA using atomic force microscopy.

The formation of G-quadruplexes in G-rich regions of DNA is believed to affect DNA transcription and replication. However, it is currently unclear how this formation occurs in the presence of a complementary strand. We have used atomic force microscopy (AFM) to image stable RNA/DNA hybrid loops generated by transcription of the plasmid pPH600, which contains a 604-bp fragment of the murine immunoglobulin Sgamma3 switch region. We show that the non-RNA-containing portion folds into G-quadruplexes, consistent with computational predictions. We also show that hybrid formation prevents further transcription from occurring, implying a regulatory role. After in vitro transcription, almost all (93%) of the plasmids had an asymmetric loop, a large asymmetric blob or a spur-like projection at the appropriate position on the DNA contour. The loops disappeared following treatment of the transcribed plasmid with RNase H, which removes mRNA hybridized with the template strand. Replacement of K+ in the transcription buffer with either Na+ or Li+ caused a reduction in the percentage of plasmids containing loops, blobs or spurs, consistent with the known effects of monovalent cations on G-quadruplex stability. The minimal sample preparation required for AFM imaging has permitted direct observation of the structural changes resulting from G-quadruplex formation.

Atomic force microscopy of the EcoKI Type I DNA restriction enzyme bound to DNA shows enzyme dimerization and DNA looping.

Atomic force microscopy (AFM) allows the study of single protein-DNA interactions such as those observed with the Type I Restriction-Modification systems. The mechanisms employed by these systems are complicated and understanding them has proved problematic. It has been known for years that these enzymes translocate DNA during the restriction reaction, but more recent AFM work suggested that the archetypal EcoKI protein went through an additional dimerization stage before the onset of translocation. The results presented here extend earlier findings confirming the dimerization. Dimerization is particularly common if the DNA molecule contains two EcoKI recognition sites. DNA loops with dimers at their apex form if the DNA is sufficiently long, and also form in the presence of ATPgammaS, a non-hydrolysable analogue of the ATP required for translocation, indicating that the looping is on the reaction pathway of the enzyme. Visualization of specific DNA loops in the protein-DNA constructs was achieved by improved sample preparation and analysis techniques. The reported dimerization and looping mechanism is unlikely to be exclusive to EcoKI, and offers greater insight into the detailed functioning of this and other higher order assemblies of proteins operating by bringing distant sites on DNA into close proximity via DNA looping.

Direct visualization of the trimeric structure of the ASIC1a channel, using AFM imaging.

There has been confusion about the subunit stoichiometry of the degenerin family of ion channels. Recently, a crystal structure of acid-sensing ion channel (ASIC) 1a revealed that it assembles as a trimer. Here, we used atomic force microscopy (AFM) to image unprocessed ASIC1a bound to mica. We detected a mixture of subunit monomers, dimers and trimers. In some cases, triple-subunit clusters were clearly visible, confirming the trimeric structure of the channel, and indicating that the trimer sometimes disaggregated after adhesion to the mica surface. This AFM-based technique will now enable us to determine the subunit arrangement within heteromeric ASICs.

Synaptotagmin perturbs the structure of phospholipid bilayers.

Synaptotagmin I (syt), an integral protein of the synaptic vesicle membrane, is believed to act as a Ca2+ sensor for neuronal exocytosis. Syt's cytoplasmic domain consists largely of two C2 domains, C2A and C2B. In response to Ca2+ binding, the C2 domains interact with membranes, becoming partially embedded in the lipid bilayer. We have imaged syt C2AB in association with lipid bilayers under fluid, using AFM. As expected, binding of C2AB to bilayers required both an anionic phospholipid [phosphatidylserine (PS)] and Ca2+. C2AB associated with bilayers in the form of aggregates of varying stoichiometries, and aggregate size increased with an increase in PS content. Repeated scanning of bilayers revealed that as C2AB dissociated it left behind residual indentations in the bilayer. The mean depth of these identations was 1.81 nm, indicating that they did not span the bilayer. Individual C2 domains (C2A and C2B) also formed aggregates and produced bilayer indentations. Binding of C2AB to bilayers and the formation of indentations were significantly compromised by mutations that interfere with binding of Ca2+ to syt or reduce the positive charge on the surface of C2B. We propose that bilayer perturbation by syt might be significant with respect to its ability to promote membrane fusion.

Determination of the architecture of ionotropic receptors using AFM imaging.

Fast neurotransmission in the nervous system is mediated by ionotropic receptors, all of which contain several subunits surrounding an integral ion channel. There are three major families of ionotropic receptors: the 'Cys-loop' receptors (including the nicotinic receptor for acetylcholine, the 5-HT(3) receptor, the GABA(A) receptor and the glycine receptor), the glutamate receptors (including the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, kainate and N-methyl-D: -aspartic acid receptors) and the P2X receptors for adenosine triphosphate. These receptors are often built from multiple types of subunit, raising the question of the stoichiometry and subunit arrangement within the receptors. This question is of therapeutic significance because in some cases drug-binding sites are located at subunit-subunit interfaces. In this paper, we describe a general method, based on atomic force microscopy imaging, to solve the architecture of multi-subunit proteins, such as the ionotropic receptors. Specific epitope tags are engineered onto each receptor subunit. The subunits are then expressed exogenously in cultured cells, and the receptors are isolated from detergent extracts of membrane fractions by affinity chromatography. The receptors are imaged both alone and in complex with anti-epitope antibodies. The size of the imaged particles provides an estimate of the subunit stoichiometry, whereas the geometry of the receptor-antibody complexes produces more detailed information about the receptor architecture. We use an automated, unbiased system to identify receptors and receptor-antibody complexes and to determine the geometry of the complexes. We are also able to determine the orientation of the receptors on the mica substrate, which will allow us to solve the subunit arrangement within receptors, such as the GABA(A) receptor, which contain three types of subunits.

Automated analysis of the architecture of receptors, imaged by atomic force microscopy.

Fast neurotransmission involves the operation of ionotropic receptors, which are multi-subunit proteins that respond to activation by opening an integral ion channel. Examples of such channels include the GABA(A) receptor, the 5-HT(3) receptor and the P2X receptor for ATP. These receptors contain more than one type of subunit, although the exact subunit stoichiometry and arrangement around the receptor rosette is often unknown. We are using atomic force microscopy (AFM) of purified receptors to address these issues. Measurement of the molecular volume of the receptor permits the determination of the number of subunits that it contains. Furthermore, analysis of the geometry of complexes between receptors and subunit-specific antibodies reveals the subunit arrangement. Our AFM-based approach has so far been dependent on manual data processing, which is both time-consuming and prone to operator bias. In this study, we set out to develop a novel method capable of automatic segmentation and quantitative analysis of both single receptor particles and receptor-antibody complexes. The method was validated using images of wild type and mutant forms of the P2X(6) receptor. We suggest that the automated method will greatly facilitate further progress in the use of AFM for the determination of receptor and multi-protein architecture.