Programmable motion of DNA origami mechanisms

DNA origami enables the precise fabrication of nanoscale geometries. We demonstrate an approach to engineer complex and reversible motion of nanoscale DNA origami machine elements. We first design, fabricate, and characterize the mechanical behavior of flexible DNA origami rotational and linear joints that integrate stiff double-stranded DNA components and flexible single-stranded DNA components to constrain motion along a single degree of freedom and demonstrate the ability to tune the flexibility and range of motion. Multiple joints with simple 1D motion were then integrated into higher order mechanisms. One mechanism is a crank–slider that couples rotational and linear motion, and the other is a Bennett linkage that moves between a compacted bundle and an expanded frame configuration with a constrained 3D motion path. Finally, we demonstrate distributed actuation of the linkage using DNA input strands to achieve reversible conformational changes of the entire structure on ∼minute timescales. Our results demonstrate programmable motion of 2D and 3D DNA origami mechanisms constructed following a macroscopic machine design approach.The ability to control, manipulate, and organize matter at the nanoscale has demonstrated immense potential for advancements in industrial technology, medicine, and materials (1–3). Bottom-up self-assembly has become a particularly promising area for nanofabrication (4, 5); however, to date designing complex motion at the nanoscale remains a challenge (6–9). Amino acid polymers exhibit well-defined and complex dynamics in natural systems and have been assembled into designed structures including nanotubes, sheets, and networks (10–12), although the complexity of interactions that govern amino acid folding make designing complex geometries extremely challenging. DNA nanotechnology, on the other hand, has exploited well-understood assembly properties of DNA to create a variety of increasingly complex designed nanostructures (13–15).Scaffolded DNA origami, the process of folding a long single-stranded DNA (ssDNA) strand into a custom structure (16–18), has enabled the fabrication of nanoscale objects with unprecedented geometric complexity that have recently been implemented in applications such as containers for drug delivery (19, 20), nanopores for single-molecule sensing (21–23), and templates for nanoparticles (24, 25) or proteins (26–28). The majority of these and other applications of DNA origami have largely focused on static structures. Natural biomolecular machines, in contrast, have a rich diversity of functionalities that rely on complex but well-defined and reversible conformational changes. Currently, the scope of biomolecular nanotechnology is limited by an inability to achieve similar motion in designed nanosystems.DNA nanotechnology has enabled critical steps toward that goal starting with the work of Mao et al. (29), who developed a DNA nanostructure that took advantage of the B–Z transition of DNA to switch states. Since then, efforts to fabricate dynamic DNA systems have primarily focused on strand displacement approaches (30) mainly on systems comprising a few strands or arrays of strands undergoing ∼nm-scale motions (31–37) in some cases guided by DNA origami templates (38–40). More recently, strand displacement has been used to reconfigure DNA origami nanostructures, for example opening DNA containers (19, 41, 42), controlling molecular binding (43, 44), or reconfiguring structures (45). The largest triggerable structural change was achieved by Han et al. in a DNA origami Möbius strip (one-sided ribbon structure) that could be opened to approximately double in size (45). Constrained motion has been achieved in systems with rotational motion (19, 20, 32, 41, 44, 46, 47) in some cases to open lid-like components (19, 20, 41) or detect molecular binding (44, 48, 49). A few of these systems achieved reversible conformational changes (32, 41, 44, 46), although the motion path and flexibility were not studied. Constrained linear motion has remained largely unexplored. Linear displacements on the scale of a few nanometers have been demonstrated via conformational changes of DNA structure motifs (50–55), strand invasion to open DNA hairpins (36, 55, 56), or the reversible sliding motion of a DNA tile actuator (56); these cases also did not investigate the motion path or flexibility of motion.Building on these prior studies, this work implements concepts from macroscopic machine design to build modular parts with constrained motion. We demonstrate an ability to tune the flexibility and range of motion and then integrate these parts into prototype mechanisms with designed 2D and 3D motion. We further demonstrate reversible actuation of a mechanism with complex conformational changes on minute timescales.     

Structure-based cleavage mechanism of Thermus thermophilus Argonaute DNA guide strand-mediated DNA target cleavage

We report on crystal structures of ternary Thermus thermophilus Argonaute (TtAgo) complexes with 5′-phosphorylated guide DNA and a series of DNA targets. These ternary complex structures of cleavage-incompatible, cleavage-compatible, and postcleavage states solved at improved resolution up to 2.2 Å have provided molecular insights into the orchestrated positioning of catalytic residues, a pair of Mg²⁺ cations, and the putative water nucleophile positioned for in-line attack on the cleavable phosphate for TtAgo-mediated target cleavage by a RNase H-type mechanism. In addition, these ternary complex structures have provided insights into protein and DNA conformational changes that facilitate transition between cleavage-incompatible and cleavage-compatible states, including the role of a Glu finger in generating a cleavage-competent catalytic Asp-Glu-Asp-Asp tetrad. Following cleavage, the seed segment forms a stable duplex with the complementary segment of the target strand.Argonaute (Ago) proteins, critical components of the RNA-induced silencing complex, play a key role in guide strand-mediated target RNA recognition, cleavage, and product release (reviewed in refs. 1–3). Ago proteins adopt a bilobal scaffold composed of an amino terminal PAZ-containing lobe (N and PAZ domains), a carboxyl-terminal PIWI-containing lobe (Mid and PIWI domains), and connecting linkers L1 and L2. Ago proteins bind guide strands whose 5′-phosphorylated and 3′-hydroxyl ends are anchored within Mid and PAZ pockets, respectively (4–7), with the anchored guide strand then serving as a template for pairing with the target strand (8, 9). The cleavage activity of Ago resides in the RNase H fold adopted by the PIWI domain (10, 11), whereby the enzyme’s Asp-Asp-Asp/His catalytic triad (12–15) initially processes loaded double-stranded siRNAs by cleaving the passenger strand and subsequently processes guide-target RNA duplexes by cleaving the target strand (reviewed in refs. 16–18). Such Mg²⁺ cation-mediated endonucleolytic cleavage of the target RNA strand (19, 20) resulting in 3′-OH and 5′-phosphate ends (21) requires Watson–Crick pairing of the guide and target strands spanning the seed segment (positions 2–2′ to 8–8′) and the cleavage site (10′–11′ step on the target strand) (9). Insights into target RNA recognition and cleavage have emerged from structural (9), chemical (22), and biophysical (23) experiments.Notably, bacterial and archaeal Ago proteins have recently been shown to preferentially bind 5′-phosphoryated guide DNA (14, 15) and use an activated water molecule as the nucleophile (reviewed in ref. 24) to cleave both RNA and DNA target strands (9). Structural studies have been undertaken on bacterial and archaeal Ago proteins in the free state (10, 15) and bound to a 5′-phosphorylated guide DNA strand (4) and added target RNA strand (8, 9). The structural studies of Thermus thermophilus Ago (TtAgo) ternary complexes have provided insights into the nucleation, propagation, and cleavage steps of target RNA silencing in a bacterial system (9). These studies have highlighted the conformational transitions on proceeding from Ago in the free state to the binary complex (4) to the ternary complexes (8, 9) and have emphasized the requirement for a precisely aligned Asp-Asp-Asp triad and a pair of Mg²⁺ cations for cleavage chemistry (9), typical of RNase H fold-mediated enzymes (24, 25). Structural studies have also been extended to binary complexes of both human (5, 6) and yeast (7) Agos bound to 5′-phosphorylated guide RNA strands.Despite these singular advances in the structural biology of RNA silencing, further progress was hampered by the modest resolution (2.8- to 3.0-Å resolution) of TtAgo ternary complexes with guide DNA (4) and added target RNAs (8, 9). This precluded identification of water molecules coordinated with the pair of Mg²⁺ cations, including the key water that acts as a nucleophile and targets the cleavable phosphate between positions 10′-11′ on the target strand. We have now extended our research to TtAgo ternary complexes with guide DNA and target DNA strands, which has permitted us to grow crystals of ternary complexes that diffract to higher (2.2–2.3 Å) resolution in the cleavage-incompatible, cleavage-compatible, and postcleavage steps. These high-resolution structures of TtAgo ternary complexes provide snapshots of distinct key steps in the catalytic cleavage pathway, opening opportunities for experimental probing into DNA target cleavage as a defense mechanism against plasmids and possibly other mobile elements (26, 27).     

Bacterial scaffold directs pole-specific centromere segregation

Bacteria use partitioning systems based on the ParA ATPase to actively mobilize and spatially organize molecular cargoes throughout the cytoplasm. The bacterium Caulobacter crescentus uses a ParA-based partitioning system to segregate newly replicated chromosomal centromeres to opposite cell poles. Here we demonstrate that the Caulobacter PopZ scaffold creates an organizing center at the cell pole that actively regulates polar centromere transport by the ParA partition system. As segregation proceeds, the ParB-bound centromere complex is moved by progressively disassembling ParA from a nucleoid-bound structure. Using superresolution microscopy, we show that released ParA is recruited directly to binding sites within a 3D ultrastructure composed of PopZ at the cell pole, whereas the ParB-centromere complex remains at the periphery of the PopZ structure. PopZ recruitment of ParA stimulates ParA to assemble on the nucleoid near the PopZ-proximal cell pole. We identify mutations in PopZ that allow scaffold assembly but specifically abrogate interactions with ParA and demonstrate that PopZ/ParA interactions are required for proper chromosome segregation in vivo. We propose that during segregation PopZ sequesters free ParA and induces target-proximal regeneration of ParA DNA binding activity to enforce processive and pole-directed centromere segregation, preventing segregation reversals. PopZ therefore functions as a polar hub complex at the cell pole to directly regulate the directionality and destination of transfer of the mitotic segregation machine.The bacterial cytoplasm is a complex mixture of dynamic macromolecules densely packed into a tiny compartment. Recent studies have revealed unexpected levels of organization of bacterial cytoplasmic components, including hundreds of proteins, specific lipids, mRNA molecules, and even the nucleoid itself (1). One strategy used by bacteria to generate subcellular organization of specific macromolecular complexes is active segregation by ParA-mediated molecular partitioning machines. ParA-based partitioning systems are found throughout bacteria and have been shown to spatially organize diverse macromolecular complexes to facilitate their equal distribution to progeny during cell division (2). An important question is how directionality is provided to ParA partitioning machines.One family of highly conserved ParA-based partitioning systems segregates plasmid or chromosomal centromeres to daughter cells during cell division. ParA-mediated DNA partitioning systems (Par systems) are composed of three core components: a centromeric DNA sequence parS, a site-specific DNA binding protein ParB that binds to the centromere parS sequence, and the ATPase ParA. Structural studies demonstrate that the activity of ParA is regulated by a molecular switch in which ATP-bound ParA forms dimers that bind tightly to DNA, and ParB stimulates ATP hydrolysis and release of ADP-bound ParA as monomers (3). During centromere partitioning in vivo, ATP-bound ParA assembles into a multimeric nucleoid-bound structure (4). At the centromere, ParB binds to the parS locus and nearby DNA to create a compact nucleoprotein complex (5). This ParB/parS complex binds to ParA subunits within the ParA/nucleoid structure, stimulating ATP hydrolysis and release of ParA-ADP (6–8). The multivalent ParB/parS complex has thus been proposed to bind to and shorten the ParA superstructure on the nucleoid, moving along a receding track via a dynamic disassembly mechanism (6, 8–10). The result of this process is the movement of the chromosomal centromere (parS) relative to the nucleoid bulk, and therefore to the cell itself.Whereas the fundamental operating principles of ParA-mediated movement seem conserved, how these machines target transfer to specific subcellular destinations is unknown. Many chromosomal Par systems maintain a single origin-proximal ParB/parS complex at the old cell pole and, after replication, move one newly replicated parS locus to the opposite pole (9, 11, 12). Polar protein complexes that interact with chromosome segregation factors have been identified in various bacteria, but the mechanistic consequences of these interactions have not been established (13–15). In Caulobacter, two distinct polar protein factors affect ParA-mediated centromere segregation: the new pole-specific protein TipN (16, 17) and the polar organizing protein PopZ (18, 19). TipN is a large, membrane-anchored, coiled-coil rich protein that localizes to the new pole throughout the cell cycle and, in addition to roles in localization of flagellar synthesis (16, 17), affects processive parS segregation via an unknown mechanism (6, 20).In contrast, PopZ is a small, acidic protein that forms a polymeric network at the cell pole (18, 19). In the prereplicative cell, PopZ localizes exclusively at the old cell pole, where it anchors the ParB-bound parS locus via direct interactions with ParB (18, 19). During chromosome replication initiation, PopZ releases ParB from the old pole and adopts a bipolar PopZ distribution that seems to capture ParB/parS complexes during the segregation process (18, 19). Whereas cells lacking tipN are only mildly elongated, popZ deletion causes severe filamentation (16–19), suggesting that PopZ plays a more important role in the regulation of segregation. However, the molecular mechanism by which PopZ affects segregation has remained elusive.Here we demonstrate that the multifunctional PopZ complex plays a crucial role in pole-directed movement of ParA-mediated chromosome segregation by interacting directly with ParA. We show that PopZ, but not TipN, is required for robust polar recruitment of ParA and demonstrate that a polar PopZ scaffold recruits and concentrates free ParA released during segregation. Recruitment of ParA within the PopZ matrix sequesters free ParA and locally regenerates ParA DNA binding activity. Active ParA complexes are released for recycling into nucleoid-bound structures near the cell pole, which we propose drives centromere segregation toward pole-localized PopZ. Thus, PopZ orchestrates a positive feedback mechanism that forces ParA-mediated centromere transfer to the cell pole. The polar PopZ scaffold complex creates a unique 3D microenvironment at the pole that spatially separates distinct centromere tethering and ParA-modulation activities, enabling coupling between chromosome segregation with the initiation of cell division.     

Insights into ParB spreading from the complex structure of Spo0J and parS

Spo0J (stage 0 sporulation protein J, a member of the ParB superfamily) is an essential component of the ParABS (partition system of ParA, ParB, and parS)-related bacterial chromosome segregation system. ParB (partition protein B) and its regulatory protein, ParA, act cooperatively through parS (partition S) DNA to facilitate chromosome segregation. ParB binds to chromosomal DNA at specific parS sites as well as the neighboring nonspecific DNA sites. Various ParB molecules can associate together and spread along the chromosomal DNA. ParB oligomer and parS DNA interact together to form a high-order nucleoprotein that is required for the loading of the structural maintenance of chromosomes proteins onto the chromosome for chromosomal DNA condensation. In this report, we characterized the binding of parS and Spo0J from Helicobacter pylori (HpSpo0J) and solved the crystal structure of the C-terminal domain truncated protein (Ct-HpSpo0J)-parS complex. Ct-HpSpo0J folds into an elongated structure that includes a flexible N-terminal domain for protein–protein interaction and a conserved DNA-binding domain for parS binding. Two Ct-HpSpo0J molecules bind with one parS. Ct-HpSpo0J interacts vertically and horizontally with its neighbors through the N-terminal domain to form an oligomer. These adjacent and transverse interactions are accomplished via a highly conserved arginine patch: RRLR. These interactions might be needed for molecular assembly of a high-order nucleoprotein complex and for ParB spreading. A structural model for ParB spreading and chromosomal DNA condensation that lead to chromosome segregation is proposed.The integrity of chromosomes and plasmids relies on precise DNA replication and segregation (1, 2). The initiation of DNA replication has to synchronize with the cell cycle to ensure precise chromosome segregation (3). In bacteria, the chromosome-encoded plasmid-partitioning system (Par) (4) and the structural maintenance of chromosomes (SMC) condensation complex (5) are two highly conserved systems associate with chromosome segregation and organization. SMC contributes to the overall stability and organization of genome (6–8). The partition system denoted ParABS is comprised of two proteins (ParA and ParB) and a centromere-like DNA element (parS) (9). ParB binds specifically to parS to form a complex. After binding ATP, ParA can interact with the ParB–parS complex to form a nucleoid–adaptor complex. ParB promotes the ATP hydrolysis activity of the complex to separate the chromosomes (9–13).In the bacterial chromosomal ParABS system, ParB has two functions: one is to regulate chromosome replication and sporulation (8, 12, 14) and the other is to participate in chromosome segregation (5, 15–17). ParB spreads along the chromosomal DNA by binding at specific parS and nonspecific DNA sites to form a high-order partition complex (18–20). This partition complex is required for the loading of SMC onto the chromosomal DNA (5). In addition, the N-terminal domain of ParB can interact with ParA and stimulate its ATPase activity (21). This nucleoid–adaptor complex, ParA–ParB–parS is used to drive chromosome segregation (22, 23). However, the detailed mechanism for this process is still unclear.Members of the ParB superfamily share similar functional domains: an N-terminal domain for protein–protein interactions, a central DNA-binding domain for parS binding, and a C-terminal domain for ParB dimerization (24). Two conserved N-terminal domain residues, Lys3 and Lys7, in the ParB from Bacillus subtilis (BsSpo0J), have been shown to interact with its regulatory protein BsSoj, a member of the ParA superfamily (3). The loss-of-function BsSpo0J R80A mutant was originally discovered by Autret et al. (25) and reportedly has disrupted focus formation by fluorescence microscopy. More recently, Graham et al. (20) showed that BsSpo0J bridges chromosomal DNA using single-molecule experiments. However, its R79A, R80A, and R82A mutants could not spread in vivo and did not bridge DNA in vitro. These highly conserved arginine residues were defined as an arginine patch (20). Furthermore, Broedersz et al. (26) studied the condensation and localization of ParB by computational simulation.The crystal structures of ParB superfamily proteins have been reported for a DNA-free form of TtSpo0J (from Thermus thermophilus, containing the N-terminal and the DNA-binding domains) (10) and three complexes: the RP4–KorB-O_B complex (from plasmid RP4, containing the DNA-binding domain) (27), the P1 ParB–parS complex (from Enterobacteria phage P1, containing the DNA-binding and the C-terminal domains) (28), and the F-SopB–sopC complex (from plasmid F, containing the DNA-binding domain) (29).The Helicobacter pylori ParABS system consists of HpSoj (ParA), HpSpo0J (ParB), and parS DNA (30, 31). Herein, we report the crystal structure of a C-terminal domain truncated HpSpo0J (Ct-HpSpo0J)–parS complex. The N-terminal and the DNA-binding domains are present on Ct-HpSpo0J. The structural details of the complex in combination with results from EMSAs, fluorescence anisotropy assay, and small angle X-ray scattering (SAXS) allow us to propose a model for ParB spreading as it relates to chromosome segregation.     

Spontaneous slow replication fork progression elicits mitosis alterations in homologous recombination-deficient mammalian cells

Homologous recombination deficient (HR⁻) mammalian cells spontaneously display reduced replication fork (RF) movement and mitotic extra centrosomes. We show here that these cells present a complex mitotic phenotype, including prolonged metaphase arrest, anaphase bridges, and multipolar segregations. We then asked whether the replication and the mitotic phenotypes are interdependent. First, we determined low doses of hydroxyurea that did not affect the cell cycle distribution or activate CHK1 phosphorylation but did slow the replication fork movement of wild-type cells to the same level than in HR⁻ cells. Remarkably, these low hydroxyurea doses generated the same mitotic defects (and to the same extent) in wild-type cells as observed in unchallenged HR⁻ cells. Reciprocally, supplying nucleotide precursors to HR⁻ cells suppressed both their replication deceleration and mitotic extra centrosome phenotypes. Therefore, subtle replication stress that escapes to surveillance pathways and, thus, fails to prevent cells from entering mitosis alters metaphase progression and centrosome number, resulting in multipolar mitosis. Importantly, multipolar mitosis results in global unbalanced chromosome segregation involving the whole genome, even fully replicated chromosomes. These data highlight the cross-talk between chromosome replication and segregation, and the importance of HR at the interface of these two processes for protection against general genome instability.DNA is continuously subjected to injury by exogenous and endogenous sources. The faithful transmission of genetic material relies on the DNA damage response (DDR), which coordinates a network of pathways, including DNA replication-repair-recombination, the cell cycle checkpoint, and chromosome segregation. A defect in any of these pathways causes genetic instability and cancer predisposition. Strikingly, both spontaneous DDR activation as a consequence of endogenous replication stress and centrosome abnormalities, which cause uneven chromosome segregation, have been reported in precancerous and early-stage malignancies (1–10). Therefore, endogenous stresses must play a key role in spontaneous chromosome instability and in cancer etiology.Homologous recombination (HR) is an evolutionarily conserved process that controls the balance between genetic stability and diversity. Specifically, HR plays a pivotal role in the reactivation of replication forks that have been blocked, contributing to DNA replication accuracy (11–16). Replication forks are routinely inactivated by endogenous stress (17, 18); therefore, HR should play an essential role to protect cells against these types of stresses, and HR deficiency should reveal endogenous replication stress. Remarkably, unchallenged HR-deficient (HR⁻) cells display both a genome-wide decrease in replication fork speed (19) and a spontaneous increase in the frequency of cells containing extra centrosomes (20–28). Two hypotheses may account for these two phenotypes. First, replication stress leads to chromosome alteration at incomplete replicated regions and chromosome rearrangements (29). However, centrosomes do not contain DNA, and if extra centrosomes at mitosis [mitotic extra centrosome (MEC)] are active, unbalanced chromosome segregation should lead to global chromosome instability, even for fully replicated chromosomes. Second, HR proteins are associated with supernumerary centrosomes; therefore, centrosome duplication defects may directly result from HR misregulation (30, 31).In this study, we addressed whether spontaneous MECs result from slow replication fork movement in HR⁻ cells. The data presented here underline the importance of HR at the molecular interface between replication and chromosome segregation to protect against spontaneous genomic instability.     

Self-assembly of alkynylplatinum(II) terpyridine amphiphiles into nanostructures via steric control and metal–metal interactions

A series of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing the hydrophilic oligo(para-phenylene ethynylene) with two 3,6,9-trioxadec-1-yloxy chains was designed and synthesized. The mononuclear alkynylplatinum(II) terpyridine complex was found to display a very strong tendency toward the formation of supramolecular structures. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would lead to the formation of nanotubes or helical ribbons. These desirable nanostructures were found to be governed by the steric bulk on the platinum(II) terpyridine moieties, which modulates the directional metal−metal interactions and controls the formation of nanotubes or helical ribbons. Detailed analysis of temperature-dependent UV-visible absorption spectra of the nanostructured tubular aggregates also provided insights into the assembly mechanism and showed the role of metal−metal interactions in the cooperative supramolecular polymerization of the amphiphilic platinum(II) complexes.Square-planar d⁸ platinum(II) polypyridine complexes have long been known to exhibit intriguing spectroscopic and luminescence properties (1–54) as well as interesting solid-state polymorphism associated with metal−metal and π−π stacking interactions (1–14, 25). Earlier work by our group showed the first example, to our knowledge, of an alkynylplatinum(II) terpyridine system [Pt(tpy)(C ≡ CR)]⁺ that incorporates σ-donating and solubilizing alkynyl ligands together with the formation of Pt···Pt interactions to exhibit notable color changes and luminescence enhancements on solvent composition change (25) and polyelectrolyte addition (26). This approach has provided access to the alkynylplatinum(II) terpyridine and other related cyclometalated platinum(II) complexes, with functionalities that can self-assemble into metallogels (27–31), liquid crystals (32, 33), and other different molecular architectures, such as hairpin conformation (34), helices (35–38), nanostructures (39–45), and molecular tweezers (46, 47), as well as having a wide range of applications in molecular recognition (48–52), biomolecular labeling (48–52), and materials science (53, 54). Recently, metal-containing amphiphiles have also emerged as a building block for supramolecular architectures (42–44, 55–59). Their self-assembly has always been found to yield different molecular architectures with unprecedented complexity through the multiple noncovalent interactions on the introduction of external stimuli (42–44, 55–59).Helical architecture is one of the most exciting self-assembled morphologies because of the uniqueness for the functional and topological properties (60–69). Helical ribbons composed of amphiphiles, such as diacetylenic lipids, glutamates, and peptide-based amphiphiles, are often precursors for the growth of tubular structures on an increase in the width or the merging of the edges of ribbons (64, 65). Recently, the optimization of nanotube formation vs. helical nanostructures has aroused considerable interests and can be achieved through a fine interplay of the influence on the amphiphilic property of molecules (66), choice of counteranions (67, 68), or pH values of the media (69), which would govern the self-assembly of molecules into desirable aggregates of helical ribbons or nanotube scaffolds. However, a precise control of supramolecular morphology between helical ribbons and nanotubes remains challenging, particularly for the polycyclic aromatics in the field of molecular assembly (64–69). Oligo(para-phenylene ethynylene)s (OPEs) with solely π−π stacking interactions are well-recognized to self-assemble into supramolecular system of various nanostructures but rarely result in the formation of tubular scaffolds (70–73). In view of the rich photophysical properties of square-planar d⁸ platinum(II) systems and their propensity toward formation of directional Pt···Pt interactions in distinctive morphologies (27–31, 39–45), it is anticipated that such directional and noncovalent metal−metal interactions might be capable of directing or dictating molecular ordering and alignment to give desirable nanostructures of helical ribbons or nanotubes in a precise and controllable manner.Herein, we report the design and synthesis of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing hydrophilic OPEs with two 3,6,9-trioxadec-1-yloxy chains. The mononuclear alkynylplatinum(II) terpyridine complex with amphiphilic property is found to show a strong tendency toward the formation of supramolecular structures on diffusion of diethyl ether in dichloromethane or dimethyl sulfoxide (DMSO) solution. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would result in nanotubes or helical ribbons in the self-assembly process. To the best of our knowledge, this finding represents the first example of the utilization of the steric bulk of the moieties, which modulates the formation of directional metal−metal interactions to precisely control the formation of nanotubes or helical ribbons in the self-assembly process. Application of the nucleation–elongation model into this assembly process by UV-visible (UV-vis) absorption spectroscopic studies has elucidated the nature of the molecular self-assembly, and more importantly, it has revealed the role of metal−metal interactions in the formation of these two types of nanostructures.     

Retinal waves regulate afferent terminal targeting in the early visual pathway

Current models of retinogeniculate development have proposed that connectivity between the retina and the dorsal lateral geniculate nucleus (dLGN) is established by gradients of axon guidance molecules, to allow initial coarse connections, and by competitive Hebbian-like processes, to drive eye-specific segregation and refine retinotopy. Here we show that when intereye competition is eliminated by monocular enucleation, blocking cholinergic stage II retinal waves disrupts the intraeye competition-mediated expansion of the retinogeniculate projection and results in the permanent disorganization of its laminae. This disruption of stage II retinal waves also causes long-term impacts on receptive field size and fine-scale retinotopy in the dLGN. Our results reveal a novel role for stage II retinal waves in regulating retinogeniculate afferent terminal targeting by way of intraeye competition, allowing for correct laminar patterning and the even allocation of synaptic territory. These findings should contribute to answering questions regarding the role of neural activity in guiding the establishment of neural circuits.The brain employs several strategies to guide the establishment of correct neural connectivity (1, 2). It has been well recognized that the high specificity of connections between the retina and the dorsal lateral geniculate nucleus (dLGN) is established through several factors. These include gradients of axon guidance molecules that guide the initial coarse targeting of afferent terminals (3–6), and spontaneous retinal activity (retinal waves) that drives competitive processes important for the refinement and segregation of afferent terminal branches (2, 7–15).Retinal waves are spontaneous propagating bursts of correlated retinal ganglion cell (RGC) activity and have been classified into three developmental stages (1, 15). Stage II retinal waves (from here on also referred to as retinal waves) are extensively studied and have been found to be critical for the development of retinofugal pathways (1, 2, 15). They are mediated by cholinergic signaling from starburst amacrine cells onto RGCs (8, 13, 16–18) and have been hypothesized to drive the Hebbian-like remodeling of RGC afferent terminals (19, 20). Retinal waves play crucial roles in both the establishment of eye-specific segregation (8, 12, 14, 20, 21), through the removal of afferent branches from opposing putative eye-specific domains, and the refinement of afferent terminals within eye-specific laminae, which is believed to be necessary for the establishment of fine-scale retinotopy (12, 22). However, studies have suggested that retinal waves might play additional roles in the development of the retinogeniculate pathway. When retinal waves are blocked during early development, mature lamination in the adult is abnormal (23–25), while eye-specific segregation recovers (26, 27). These results uncovered a retinal wave-dependent window for the development of retinogeniculate lamination. However, the question remains open as to whether these lamination defects are due to abnormal late eye-specific segregation or the disruption of some form of retinal wave-dependent afferent terminal targeting.A potential retinal wave-dependent mechanism that could regulate retinogeniculate afferent terminal targeting is axon–axon competition originating from the same eye (i.e., intraeye competition). Classic studies in goldfish first demonstrated the principle of axon–axon competition at the optic tectum (28). These studies showed that RGC afferent terminals can undergo expansive or compressive rearrangements in their targeting in response to changes in afferent number, or retinorecipient target size, while maintaining correct retinotopy (28–32). Similarly, neonatal monocular enucleation in ferrets results in an expanded ipsilateral and contralateral projection by adulthood, while correct laminar organization is maintained (7, 10). This demonstrates that retinogeniculate afferent terminals can undergo an expansive and orderly rearrangement due to intraeye competition, and that intereye competition is not required for the establishment of proper retinogeniculate lamination.To investigate whether retinal waves play a role in regulating retinogeniculate afferent terminal targeting by way of intraeye competition, we monocularly enucleated ferrets one day after birth (P1), to eliminate intereye competition, while also pharmacologically blocking retinal waves (P1– P10) in the surviving eye with the cholinergic agonist epibatidine (EPI) (8, 13, 18). Effects on the targeting of retinogeniculate afferents terminals were assessed anatomically, to characterize impacts on retinogeniculate lamination, and functionally, to assess changes in receptive field (RF) structure and retinotopy in the dLGN. Our results demonstrate that retinal waves regulate afferent terminal targeting by way of intraeye competition during the development of the retinogeniculate pathway.     

Centromere proteins CENP-C and CAL1 functionally interact in meiosis for centromere clustering,pairing, and chromosome segregation

Meiotic chromosome segregation involves pairing and segregation of homologous chromosomes in the first division and segregation of sister chromatids in the second division. Although it is known that the centromere and kinetochore are responsible for chromosome movement in meiosis as in mitosis, potential specialized meiotic functions are being uncovered. Centromere pairing early in meiosis I, even between nonhomologous chromosomes, and clustering of centromeres can promote proper homolog associations in meiosis I in yeast, plants, and Drosophila. It was not known, however, whether centromere proteins are required for this clustering. We exploited Drosophila mutants for the centromere proteins centromere protein-C (CENP-C) and chromosome alignment 1 (CAL1) to demonstrate that a functional centromere is needed for centromere clustering and pairing. The cenp-C and cal1 mutations result in C-terminal truncations, removing the domains through which these two proteins interact. The mutants show striking genetic interactions, failing to complement as double heterozygotes, resulting in disrupted centromere clustering and meiotic nondisjunction. The cluster of meiotic centromeres localizes to the nucleolus, and this association requires centromere function. In Drosophila, synaptonemal complex (SC) formation can initiate from the centromere, and the SC is retained at the centromere after it disassembles from the chromosome arms. Although functional CENP-C and CAL1 are dispensable for assembly of the SC, they are required for subsequent retention of the SC at the centromere. These results show that integral centromere proteins are required for nuclear position and intercentromere associations in meiosis.Centromeres are the control centers for chromosomes, and thus are essential for accurate segregation in cell division. Centromeres are the DNA regions with a specialized chromatin structure upon which the kinetochore is built. The kinetochore is a complex of at least 100 proteins that contains the proteins to bind microtubules, motors to move on or destabilize microtubules, as well as checkpoint proteins monitoring kinetochore–microtubule attachment (1). The ability of the kinetochore to control microtubule binding and chromosome movement is essential for proper segregation in both mitosis and meiosis. In meiosis, additional constraints are placed on kinetochore function to ensure that homologs segregate in the first division and that segregation of sister chromatids is deferred until the second division (2). Recent studies indicate that in addition, the centromere itself may influence homolog segregation by controlling homolog pairing and formation of the synaptonemal complex (SC) (3).In prophase of meiosis I, the homologs must pair and ultimately become attached, usually by recombination and crossing-over. By quantifying centromere number through prophase I, it has been observed that centromeres pair in yeast, plants, and Drosophila (3). Perhaps unexpectedly, this pairing can be between nonhomologous centromeres; in yeast, this has been proposed as a mechanism to prevent recombination around the centromere, as centromere pairing resolves from initially being nonhomologous to being homologous (4, 5). Homologous centromere pairing may play a critical role in ensuring segregation of chromosomes that do not undergo crossing-over, possibly by affecting orientation of the kinetochores (3, 6–8).The centromere also regulates synapsis via the formation of the SC. SC formation initiates at the centromere and sites of cross-over formation in yeast, and the centromere is the first site for SC formation in Drosophila prophase I (9, 10). In addition, the SC persists at the centromere in yeast and Drosophila after the SC present along the chromosome arms has disassembled late in prophase I (7, 9, 11). Although SC assembly does not begin at centromeres in mouse meiosis, it persists at the centromeres and appears to promote proper segregation (12, 13).Another centromere property has been observed in Drosophila oocytes. In most organisms, the centromeres are clustered together at one site at the onset of meiosis, likely a remnant of their configuration in mitosis, but this clustering breaks down as centromeres arrange in pairs (3, 4). In Drosophila, however, the centromeres remain clustered until exit from prophase I at oocyte maturation (9, 10, 14). Although an essential role for centromere clustering has not been demonstrated, it may facilitate homolog pairing, synapsis, or accurate segregation, particularly given that the homologous telomeres do not pair into a bouquet formation in Drosophila meiosis (15, 16). Components of the SC are necessary for centromere clustering, as is the cohesion protein ORD (9, 14).The studies on centromere pairing and clustering define centromere geography within the meiotic nucleus, but they did not test whether centromere structure or function was involved. Centromeres have specialized nucleosomes with a histone H3 variant, centromere protein-A (CENP-A) (17). Incorporation of CENP-A into centromere chromatin is regulated precisely, although it occurs at distinct cell cycle times in different cell types, varying between late mitosis and G1 (17). In vertebrates, a complex of 15 proteins, the constitutive centromere-associated network (CCAN), is present on the CENP-A chromatin throughout the cell cycle and is crucial for assembling kinetochore proteins (1). In Drosophila, the entire CCAN complex has not been identified, although the CENP-C protein is present (18). Another Drosophila protein, CAL1, binds to CENP-A (called CID in Drosophila) in a prenucleosomal complex, and CAL1 is required for loading CID (19–22). CAL1 interacts with both CID and CENP-C, and all three proteins show interdependency for centromere localization (21, 23).Little is known about the activities of these centromere proteins in meiosis. In fission yeast, CENP-C has been demonstrated to be critical for kinetochore–microtubule binding in meiosis and also to control kinetochore orientation in meiosis I (24). The timing of assembly of kinetochore and centromere proteins onto meiotic chromosomes has been examined in mouse spermatocytes (25) and in Drosophila spermatocytes and sperm (26, 27). RNAi studies have shown that CAL1 and CENP-C (the latter to a lesser extent) are needed for CID localization in Drosophila male meiosis, with reduction in the levels of any of these three proteins being associated with meiotic segregation errors (26). Drosophila males differ from most organisms in not undergoing recombination or forming an SC, and centromere clustering does not occur (28). A question of particular interest that has yet to be addressed is whether centromere architecture and function are required for centromere clustering and pairing in meiosis.     

Ionizing irradiation-induced radical stress stalls live meiotic chromosome movements by altering the actin cytoskeleton

Meiosis generates haploid cells or spores for sexual reproduction. As a prelude to haploidization, homologous chromosomes pair and recombine to undergo segregation during the first meiotic division. During the entire meiotic prophase of the yeast Saccharomyces cerevisiae, chromosomes perform rapid movements that are suspected to contribute to the regulation of recombination. Here, we investigated the impact of ionizing radiation (IR) on movements of GFP–tagged bivalents in live pachytene cells. We find that exposure of sporulating cultures with >40 Gy (4-krad) X-rays stalls pachytene chromosome movements. This identifies a previously undescribed acute radiation response in yeast meiosis, which contrasts with its reported radioresistance of up to 1,000 Gy in survival assays. A modified 3′-end labeling assay disclosed IR-induced dsDNA breaks (DSBs) in pachytene cells at a linear dose relationship of one IR-induced DSB per cell per 5 Gy. Dihydroethidium staining revealed formation of reactive oxygen species (ROS) in irradiated cells. Immobility of fuzzy-appearing irradiated bivalents was rescued by addition of radical scavengers. Hydrogen peroxide-induced ROS did reduce bivalent mobility similar to 40 Gy X IR, while they failed to induce DSBs. IR- and H₂O₂-induced ROS were found to decompose actin cables that are driving meiotic chromosome mobility, an effect that could be rescued by antioxidant treatment. Hence, it appears that the meiotic actin cytoskeleton is a radical-sensitive system that inhibits bivalent movements in response to IR- and oxidant-induced ROS. This may be important to prevent motility-driven unfavorable chromosome interactions when meiotic recombination has to proceed in genotoxic environments.Exposure to ionizing irradiation (IR) has dire consequences for the cell, because it causes the formation of radicals and reactive oxygen species (ROS) that can oxidize and damage cellular components including proteins and DNA (1), whereas protection from IR-induced radical-mediated protein oxidation can lead to significant radio resistance (2). At the DNA level, IR leads to single-stranded and double-stranded DNA breaks (DSBs), with the latter being a severe threat to cellular survival (3). To cope with DSBs that may arise physiologically and/or by genotoxic environmental impacts such as IR, the cell repairs DSBs by two major pathways, nonhomologous end joining (NHEJ) and homologous recombination (HR), which predominate in the G1 and G2 phase of the cell cycle, respectively, and underlie cell-cycle-dependent sensitivities to IR exposure (4, 5). In the G2 phase and in the first meiotic prophase, DSB repair is mediated by HR, in yeast meiosis addressing ∼150 DSBs (6, 7) that are formed by the Topo2-related endonuclease/transesterase enzyme SPO11 (8). Absence of DSBs and the resulting compromised spore viability (9) can be partially rescued by ionizing irradiation (10). In all, yeast cells exhibit a high resistance to IR (11–13) which is also true for cells in prophase I (10, 14). In the meiosis of numerous species, programmed Spo11-induced DSBs are instrumental for homologous chromosome search and pairing and provide the substrate for HR, generating two outcomes: noncrossovers (NCO) and crossovers (CO) that allow for homolog segregation in the meiosis I division (reviewed by refs. 15 and 16). For CO to occur, homologous chromosomes need to encounter and pair lengthwise (synapse) during first meiotic prophase (see ref. 17). Homolog pairing occurs after completion of premeiotic DNA replication. Live cell studies in the synaptic meiosis of the yeast Saccharomyces cerevisiae (2n = 32) have shown that meiotic telomeres (18), chromosomes, and bivalents undergo a striking mobility throughout the entire prophase I (19–21), which contrasts with the relative immobility of pachytene bivalents in mammalian prophase I (22). It has been found that rapid and continuous telomere and chromosome movements in budding yeast meiocytes depend on actin polymerization (18–20) and an intact meiotic telomere complex (21, 23, 24). Besides a general mobility of chromosomes throughout prophase I, single bivalents are capable to rapidly move away and return to the motile chromosome mass, a behavior termed “maverick” formation (19) or rapid chromosome movements (20, 21).Exposure to ionizing radiation induces a plethora of physicochemical effects in the irradiated cells including DNA damage (1, 3). Extensive research addressing the adverse effects of IR exposure using yeast as a model system had largely been directed toward mutation induction, DSB repair, and cell cycle effects (e.g., 11–13, 25, 26). Meiotic yeast cells exposed to 50–80 krad (500–800 Gy) X or γ irradiation have been shown to exhibit a profound reduction in cell survival, particularly when exposed in the G1 cell cycle phase that lacks a sister chromatid for repair (4). Irradiated meiotic yeast cells exhibit mutations and chromosome missegregation at meiosis I, leading to reduced sporulation (5, 10, 27). While previous studies addressed late deterministic effects in irradiated yeast cells such as DNA repair, mutations, and cell survival, we were interested in the immediate consequences of IR exposure on motile meiotic chromosomes. Bivalent mobility can be expected to promote chromosomal rearrangements, if it continues after the formation of ectopic unregulated DSBs. Chromosomal translocations have, for instance, been observed after irradiation of mitotic budding yeast cells (28) and of meiotic prophase cells of mice (29). Furthermore, meiotic chromosome mobility has been proposed to be involved in regulating (adverse) chromosomal interactions (30). To study the consequences of IR exposure on meiotic chromosome mobility we followed live bivalent movements in X-irradiated and nonirradiated yeast cells expressing the GFP-tagged version of the synaptonemal complex protein ZIP1 (19) undergoing sporulation.     

WWOX,the common fragile site FRA16D gene product,regulates ATM activation and the DNA damage response

Genomic instability is a hallmark of cancer. The WW domain-containing oxidoreductase (WWOX) is a tumor suppressor spanning the common chromosomal fragile site FRA16D. Here, we report a direct role of WWOX in DNA damage response (DDR) and DNA repair. We show that Wwox deficiency results in reduced activation of the ataxia telangiectasia-mutated (ATM) checkpoint kinase, inefficient induction and maintenance of γ-H2AX foci, and impaired DNA repair. Mechanistically, we show that, upon DNA damage, WWOX accumulates in the cell nucleus, where it interacts with ATM and enhances its activation. Nuclear accumulation of WWOX is regulated by its K63-linked ubiquitination at lysine residue 274, which is mediated by the E3 ubiquitin ligase ITCH. These findings identify a novel role for the tumor suppressor WWOX and show that loss of WWOX expression may drive genomic instability and provide an advantage for clonal expansion of neoplastic cells.Genomic instability is a common characteristic of human cancers. The DNA damage response (DDR) maintains the integrity of the genome in response to DNA damage. DDR is a complex signaling process that results in cell cycle arrest followed by either DNA repair or apoptosis if the DNA damage is too extensive to be repaired (1–3). Key mammalian damage response sensors are ataxia telangiectasia-mutated (ATM), ATM and Rad3-related, and DNA-dependent PKs (4, 5). Disruption of the DDR machinery in human cells leads to genomic instability and an increased risk of cancer progression (6, 7).The WW domain-containing oxidoreductase (WWOX) gene spans the common fragile site (CFS) FRA16D (8, 9). Genomic alterations affecting the WWOX locus have been reported in several types of cancer and include homozygous and hemizygous deletions (10–13). Ectopic expression of WWOX in WWOX-negative cancer cells attenuates cell growth and suppresses tumor growth in immunocompromised mice (10, 11, 14). Importantly, targeted ablation of Wwox in mice results in higher incidence of spontaneous lesions resembling osteosarcomas and lung and mammary tumors (14–16). These findings suggest WWOX as a tumor suppressor. The WWOX protein contains two N-terminal WW domains mediating WWOX interaction with PP(proline)x(amino acid)Y(tyrosine)-containing proteins (11, 17) and a central short-chain deyhdrogenase/reductase domain that has been proposed to function in steroidogenesis (18). Recent characterization of WWOX domains revealed that they interact, mainly through the WW1 domain, with multiprotein networks (3). The mechanism by which WWOX suppresses tumorigenicity is, however, not well-known.In vitro, CFSs are defined as gaps or breaks on metaphase chromosomes that occur in cells treated with inhibitors of DNA replication (19, 20). In vivo, CFSs are preferential targets of replication stress in preneoplastic lesions (21), and emerging evidence suggests that they represent early warning sensors for DNA damage (22–24). Both genetic and epigenetic factors are thought to regulate the fragility of CFS (25, 26). Recent profiling studies of CFS provide evidence that the functional fragility of CFS is tissue-specific (27–29). High-throughput genomic analyses of 3,131 cancer specimens (12) and 746 cancer cell lines (13) have recently identified large deletions in CFSs, including the FRA16D/WWOX locus. Although these deletions have been linked to the presence of DNA replication stress (30), the molecular function of gene products of CFSs, including the WWOX protein, is poorly understood. Here, we identify a direct role of WWOX in the DDR and show that the WWOX gene product functions as a modulator of the DNA damage checkpoint kinase ATM.     

From the Cover: PNAS Plus: Single-molecule visualization of RecQ helicase reveals DNA melting,nucleation, and assembly are required for processive DNA unwinding

DNA helicases are motor proteins that unwind double-stranded DNA (dsDNA) to reveal single-stranded DNA (ssDNA) needed for many biological processes. The RecQ helicase is involved in repairing damage caused by DNA breaks and stalled replication forks via homologous recombination. Here, the helicase activity of RecQ was visualized on single molecules of DNA using a fluorescent sensor that directly detects ssDNA. By monitoring the formation and progression of individual unwinding forks, we observed that both the frequency of initiation and the rate of unwinding are highly dependent on RecQ concentration. We establish that unwinding forks can initiate internally by melting dsDNA and can proceed in both directions at up to 40–60 bp/s. The findings suggest that initiation requires a RecQ dimer, and that continued processive unwinding of several kilobases involves multiple monomers at the DNA unwinding fork. We propose a distinctive model wherein RecQ melts dsDNA internally to initiate unwinding and subsequently assembles at the fork into a distribution of multimeric species, each encompassing a broad distribution of rates, to unwind DNA. These studies define the species that promote resection of DNA, proofreading of homologous pairing, and migration of Holliday junctions, and they suggest that various functional forms of RecQ can be assembled that unwind at rates tailored to the diverse biological functions of RecQ helicase.DNA helicases are ubiquitous enzymes involved in many aspects of DNA metabolism, including DNA replication, repair, and recombination. These enzymes work by coupling the hydrolysis of nucleoside triphosphates (NTPs) to unwinding of double-stranded DNA (dsDNA) to produce single-stranded DNA (ssDNA) (1). This activity allows the cell’s machinery to access the information stored within the bases of the double helix. RecQ helicase from Escherichia coli is the founding member of the RecQ family of helicases (2). These enzymes belong to the superfamily 2 (SF2) group of helicases, yet share greater sequence homology with their own family members, and they play important roles in the maintenance of genomic integrity by DNA recombination and repair (1, 3). Mutations in the human RecQ-like helicases, Bloom (BLM), Werner (WRN), and RecQ4 proteins, lead to Bloom’s, Werner’s, and Rothmund–Thomson syndromes, respectively. These genetic disorders are characterized by genomic instability and an increased incidence of cancers (4).E. coli RecQ is a 3′ → 5′ helicase that functions in DNA-break repair by homologous recombination (2, 5, 6). RecQ and RecJ, a 5′ → 3′ exonuclease, process ssDNA gaps or dsDNA breaks into ssDNA for recombinational repair by RecA (7–10). In addition, RecQ ensures recombination fidelity in vivo by removing inappropriately paired joint molecules to prevent illegitimate recombination and also by disrupting joint molecule intermediates to facilitate repair by synthesis-dependent strand annealing, preventing chromosomal crossing over (7, 9, 11, 12). RecQ also functions with topoisomerase III (Topo III), a type I topoisomerase, to catenate and decatenate DNA molecules and to separate converged replication forks (13, 14). RecQ and Topo III provide an alternative to the RuvABC pathway for disengaging double Holliday junctions and do so without producing chromosomal crossovers (15).In vitro, RecQ can unwind a multitude of DNA substrates and does not require a ssDNA tail, or even a dsDNA end, to initiate unwinding; consequently, it is distinctive in being able to unwind covalently closed circular plasmid DNA (16, 17). The winged-helix domain of RecQ is important for the recognition of this broad array of DNA substrates (18). This domain binds to dsDNA yet it adopts a flexible conformation that allows it to adapt to many DNA structures. A curious feature of RecQ is that maximal unwinding requires nearly stoichiometric amounts of protein relative to the DNA (one protein per ∼10 bp), which can be partially mitigated (one protein per ∼30 bp) by including the ssDNA binding protein, SSB (5, 6, 16, 19). This behavior is compatible with the low processivity of ssDNA translocation (∼30–100 nucleotides) by RecQ (20–22) and other RecQ members (23). Paradoxically, at limiting concentrations, most RecQ helicases nonetheless efficiently unwind several kilobases of dsDNA in the course of their normal functions (9, 16, 24–26), suggesting a dynamic unwinding process. Although RecQ can unwind DNA as a monomer, it also shows a functional cooperativity when unwinding DNA with an ssDNA tail (27–29). Thus, multiple monomers can bind to ssDNA to unwind long dsDNA regions.Fluorescence techniques coupled with single-molecule microscopy have emerged as a powerful method for studying the unwinding mechanism of helicases (30–35). These assays measure individual enzymes directly or indirectly through their actions on individual DNA molecules, thus alleviating many of the drawbacks of ensemble experiments. In particular, total internal reflection fluorescence (TIRF) microscopy permits the detection of individual fluorophores with high sensitivity in the presence of a high background (30). To visualize the activity of DNA binding proteins and helicases, TIRF microscopy can be coupled with microfluidic techniques to facilitate visualization of molecules and exchange of solution components (36–39).Ensemble assays have elucidated many features of DNA unwinding by the RecQ helicase family, but the need to average over a heterogeneous and unsynchronized population of enzymes has precluded a thorough understanding of this diverse and universally important helicase family. To permit a more insightful analysis, we directly visualized unwinding of individual molecules of DNA by RecQ helicase. Unwinding was monitored using fluorescent SSB to visualize generation of ssDNA. On single DNA molecules, we could see tracks of the fluorescent SSB binding to ssDNA produced by the helicase activity of RecQ. We show that RecQ initiates DNA unwinding via melting of duplex DNA at internal sites. Once initiated, DNA unwinding propagates either uni- or bidirectionally via the cooperative action of multiple RecQ molecules at the junction of ssDNA with dsDNA. Collectively, these observations define a stable oligomeric complex of subunits involved in processive helicase action, which is concordant with both biochemical and biological function of RecQ helicases and other helicases.