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To perform double-stranded DNA passage, type II topoisomerases generate a covalent enzyme-cleaved DNA complex (i.e. cleavage complex). Although this complex is a requisite enzyme intermediate, it is also intrinsically dangerous to genomic stability. Consequently, cleavage complexes are the targets for several clinically relevant anticancer and antibacterial drugs. Human topoisomerase IIα and IIβ and bacterial gyrase maintain higher levels of cleavage complexes with negatively supercoiled over positively supercoiled DNA substrates. Conversely, bacterial topoisomerase IV is less able to distinguish DNA supercoil handedness. Despite the importance of supercoil geometry to the activities of type II topoisomerases, the basis for supercoil handedness recognition during DNA cleavage has not been characterized. Based on the results of benchtop and rapid-quench flow kinetics experiments, the forward rate of cleavage is the determining factor of how topoisomerase IIα/IIβ, gyrase and topoisomerase IV distinguish supercoil handedness in the absence or presence of anticancer/antibacterial drugs. In the presence of drugs, this ability can be enhanced by the formation of more stable cleavage complexes with negatively supercoiled DNA. Finally, rates of enzyme-mediated DNA ligation do not contribute to the recognition of DNA supercoil geometry during cleavage. Our results provide greater insight into how type II topoisomerases recognize their DNA substrates.

ParABS partition systems, comprising the centromere-like DNA sequence parS, the parS-binding ParB-CTPase, and the nucleoid-binding ParA-ATPase, ensure faithful segregation of bacterial chromosomes and low-copy-number plasmids. F-plasmid partition complexes containing ParBF and parSF move by generating and following a local concentration gradient of nucleoid-bound ParAF. However, the process through which ParBF activates ParAF-ATPase has not been defined. We studied CTP- and parSF-modulated ParAF–ParBF complex assembly, in which DNA-bound ParAF-ATP dimers are activated for ATP hydrolysis by interacting with two ParBF N-terminal domains. CTP or parSF enhances the ATPase rate without significantly accelerating ParAF–ParBF complex assembly. Together, parSF and CTP accelerate ParAF–ParBF assembly without further significant increase in ATPase rate. Magnetic-tweezers experiments showed that CTP promotes multiple ParBF loading onto parSF-containing DNA, generating condensed partition complex-like assemblies. We propose that ParBF in the partition complex adopts a conformation that enhances ParBF–ParBF and ParAF–ParBF interactions promoting efficient partitioning.

Next-generation sequencing (NGS) platforms have been adapted to generate genome-wide maps and sequence context of binding and cleavage of DNA topoisomerases (topos). Continuous refinements of these techniques have resulted in the acquisition of data with unprecedented depth and resolution, which has shed new light on in vivo topo behavior. Topos regulate DNA topology through the formation of reversible single- or double-stranded DNA breaks. Topo activity is critical for DNA metabolism in general, and in particular to support transcription and replication. However, the binding and activity of topos over the genome in vivo was difficult to study until the advent of NGS. Over and above traditional chromatin immunoprecipitation (ChIP)-seq approaches that probe protein binding, the unique formation of covalent protein–DNA linkages associated with DNA cleavage by topos affords the ability to probe cleavage and, by extension, activity over the genome. NGS platforms have facilitated genome-wide studies mapping the behavior of topos in vivo, how the behavior varies among species and how inhibitors affect cleavage. Many NGS approaches achieve nucleotide resolution of topo binding and cleavage sites, imparting an extent of information not previously attainable. We review the development of NGS approaches to probe topo interactions over the genome in vivo and highlight general conclusions and quandaries that have arisen from this rapidly advancing field of topoisomerase research.

Type IA topoisomerases cleave single-stranded DNA and relieve negative supercoils in discrete steps corresponding to the passage of the intact DNA strand through the cleaved strand. Although type IA topoisomerases are assumed to accomplish this strand passage via a protein-mediated DNA gate, opening of this gate has never been observed. We developed a single-molecule assay to directly measure gate opening of the Escherichia coli type IA topoisomerases I and III. We found that after cleavage of single-stranded DNA, the protein gate opens by as much as 6.6 nm and can close against forces in excess of 16 pN. Key differences in the cleavage, ligation, and gate dynamics of these two enzymes provide insights into their different cellular functions. The single-molecule results are broadly consistent with conformational changes obtained from molecular dynamics simulations. These results allowed us to develop a mechanistic model of interactions between type IA topoisomerases and single-stranded DNA. Single-molecule magnetic tweezers analyses and supporting MD simulations provide evidence for protein-gate opening of type IA topoisomerases.

Fanconi Anemia (FA) is characterized by bone marrow failure, congenital abnormalities, and cancer. Of over 20 FA-linked genes, FANCJ uniquely encodes a DNA helicase and mutations are also associated with breast and ovarian cancer. fancj−/− cells are sensitive to DNA interstrand cross-linking (ICL) and replication fork stalling drugs. We delineated the molecular defects of two FA patient-derived FANCJ helicase domain mutations. FANCJ-R707C was compromised in dimerization and helicase processivity, whereas DNA unwinding by FANCJ-H396D was barely detectable. DNA binding and ATP hydrolysis was defective for both FANCJ-R707C and FANCJ-H396D, the latter showing greater reduction. Expression of FANCJ-R707C or FANCJ-H396D in fancj−/− cells failed to rescue cisplatin or mitomycin sensitivity. Live-cell imaging demonstrated a significantly compromised recruitment of FANCJ-R707C to laser-induced DNA damage. However, FANCJ-R707C expressed in fancj-/- cells conferred resistance to the DNA polymerase inhibitor aphidicolin, G-quadruplex ligand telomestatin, or DNA strand-breaker bleomycin, whereas FANCJ-H396D failed. Thus, a minimal threshold of FANCJ catalytic activity is required to overcome replication stress induced by aphidicolin or telomestatin, or to repair bleomycin-induced DNA breakage. These findings have implications for therapeutic strategies relying on DNA cross-link sensitivity or heightened replication stress characteristic of cancer cells.

Gyrase and topoisomerase IV are the targets of fluoroquinolone antibacterials. However, the rise in antimicrobial resistance has undermined the clinical use of this important drug class. Therefore, it is critical to identify new agents that maintain activity against fluoroquinolone-resistant strains. One approach is to develop non-fluoroquinolone drugs that also target gyrase and topoisomerase IV, but interact differently with the enzymes. This has led to the development of the “novel bacterial topoisomerase inhibitor” (NBTI) class of antibacterials. Despite the clinical potential of NBTIs, there is a relative paucity of data describing their mechanism of action against bacterial type II topoisomerases. Consequently, we characterized the activity of GSK126, a naphthyridone/aminopiperidine-based NBTI, against a variety of Gram-positive and Gram-negative bacterial type II topoisomerases including gyrase from Mycobacterium tuberculosis, and gyrase and topoisomerase IV from Bacillus anthracis and Escherichia coli. GSK126 enhanced single-stranded DNA cleavage and suppressed double-stranded cleavage mediated by these enzymes. It was also a potent inhibitor of gyrase-catalyzed DNA supercoiling and topoisomerase IV-catalyzed decatenation. Thus, GSK126 displays a similar bimodal mechanism of action across a variety of species. In contrast, GSK126 displayed a variable ability to overcome fluoroquinolone resistance mutations across these same species. Our results suggest that NBTIs elicit their antibacterial effects by two different mechanisms: inhibition of gyrase/topoisomerase IV catalytic activity or enhancement of enzyme-mediated DNA cleavage. Furthermore, the relative importance of these two mechanisms appears to differ from species to species. Therefore, we propose that the mechanistic basis for the antibacterial properties of NBTIs is bimodal in nature.

In magnetic tweezers experiments, we observe that torsional DNA buckling rates and transition state distances are insensitive to base-pairing defects. This is surprising because defects are expected to kink DNA and lower the energy of a localized loop. Nonetheless, base-pairing defects lead to pinning of buckled structures at the defects, which may be important for DNA repair in vivo. We find that the decrease in entropy from pinning roughly balances the decrease in bending energy, explaining why defects have little effect on buckling rates. Our data are generally consistent with elastic rod theory, which predicts that the transition-state structure for torsional buckling is a localized wave with a specific shape (“soliton”). The transition-state soliton decays to a metastable looped intermediate (“curl”) that is separated from the final, fully-buckled state by a second, low energy, barrier. DNAs with base mismatch defects buckle at lower torque, where elastic rod theory predicts the loop structure is more stable, and manifest an intermediate buckling structure consistent with such a loop. We estimate that, under our high force, high salt experimental conditions, the soliton barrier is approximately 10 k(B)T and, to reach this transition state from the unbuckled state, the system torque instantaneously decreases by approximately 1 pN·nm for DNA with or without a small defect.

DNA segregation is a critical process for all life, and although there is a relatively good understanding of eukaryotic mitosis, the mechanism in bacteria remains unclear. The small size of a bacterial cell and the number of factors involved in its subcellular organization make it difficult to study individual systems under controlled conditions in vivo. We developed a cell-free technique to reconstitute and visualize bacterial ParA-mediated segregation systems. Our studies provide direct evidence for a mode of transport that does not use a classical cytoskeletal filament or motor protein. Instead, we demonstrate that ParA-type DNA segregation systems can establish a propagating ParA ATPase gradient on the nucleoid surface, which generates the force required for the directed movement of spatially confined cargoes, such as plasmids or large organelles, and distributes multiple cargos equidistant to each other inside cells. Here we present the critical principles of our diffusion-ratchet model of ParA-mediated transport and expand on the mathematically derived chemophoresis force using experimentally-determined biochemical and cellular parameters.

Bacterial plasmids are extrachromosomal DNA that provides selective advantages for bacterial survival. Plasmid partitioning can be remarkably robust. For high-copy-number plasmids, diffusion ensures that both daughter cells inherit plasmids after cell division. In contrast, most low-copy-number plasmids need to be actively partitioned by a conserved tripartite ParA-type system. ParA is an ATPase that binds to chromosomal DNA; ParB is the stimulator of the ParA ATPase and specifically binds to the plasmid at a centromere-like site, parS. ParB stimulation of the ParA ATPase releases ParA from the bacterial chromosome, after which it takes a long time to reset its DNA-binding affinity. We previously demonstrated in vitro that the ParA system can exploit this biochemical asymmetry for directed cargo transport. Multiple ParA-ParB bonds can bridge a parS-coated cargo to a DNA carpet, and they can work collectively as a Brownian ratchet that directs persistent cargo movement with a ParA-depletion zone trailing behind. By extending this model, we suggest that a similar Brownian ratchet mechanism recapitulates the full range of actively segregated plasmid motilities observed in vivo. We demonstrate that plasmid motility is tuned as the replenishment rate of the ParA-depletion zone progressively increases relative to the cargo speed, evolving from diffusion to pole-to-pole oscillation, local excursions, and, finally, immobility. When the plasmid replicates, the daughters largely display motilities similar to that of their mother, except that when the single-focus progenitor is locally excursive, the daughter foci undergo directed segregation. We show that directed segregation maximizes the fidelity of plasmid partition. Given that local excursion and directed segregation are the most commonly observed modes of plasmid motility in vivo, we suggest that the operation of the ParA-type partition system has been shaped by evolution for high fidelity of plasmid segregation.

Magnetic tweezers is a versatile and easy to implement single-molecule technique that has become increasingly prevalent in the study of nucleic acid based molecular motors. Here, we provide a description of the magnetic tweezers instrument and guidelines for measuring and analyzing DNA helicase activity. Along with experimental methods, we describe a robust method of single-molecule trajectory analysis based on the Student's t-test that accommodates continuous transitions in addition to the discrete transitions assumed in most widely employed analysis routines. To illustrate the single-molecule unwinding assay and the analysis routine, we provide DNA unwinding measurements of Escherichia coli RecQ helicase under a variety of conditions (Na+, ATP, temperature, and DNA substrate geometry). These examples reveal that DNA unwinding measurements under various conditions can aid in elucidating the unwinding mechanism of DNA helicase but also emphasize that environmental effects on DNA helicase activity must be considered in relation to in vivo activity and mechanism.

Type IIA topoisomerases modify DNA topology by passing one segment of duplex DNA (transfer or T–segment) through a transient double-strand break in a second segment of DNA (gate or G–segment) in an ATP-dependent reaction. Type IIA topoisomerases decatenate, unknot and relax supercoiled DNA to levels below equilibrium, resulting in global topology simplification. The mechanism underlying this non-equilibrium topology simplification remains speculative. The bend angle model postulates that non-equilibrium topology simplification scales with the bend angle imposed on the G–segment DNA by the binding of a type IIA topoisomerase. To test this bend angle model, we used atomic force microscopy and single-molecule Forster resonance energy transfer to measure the extent of bending imposed on DNA by three type IIA topoisomerases that span the range of topology simplification activity. We found that Escherichia coli topoisomerase IV, yeast topoisomerase II and human topoisomerase IIα each bend DNA to a similar degree. These data suggest that DNA bending is not the sole determinant of non-equilibrium topology simplification. Rather, they suggest a fundamental and conserved role for DNA bending in the enzymatic cycle of type IIA topoisomerases.

Type IIA Topoisomerases are essential enzymes that contribute to chromosomal integrity by controlling the degree of supercoiling and segregating newly replicated chromosomes1. They share a conserved core mechanism in which a double-stranded segment of DNA is passed through a transient double-stranded break in a second segment of DNA (Figure 1). Remarkably, this conserved strand passage reaction results in a range of distinct activities catalyzed by different but related type IIA topoisomerases2. At one extreme is prokaryotic DNA gyrase that negatively supercoils DNA, but is a poor decatenase. At the other extreme is the closely related prokaryotic topoisomerase IV that is an efficient decatenase and relaxes positive supercoils more efficiently than negative supercoils1. Eukaryotic type IIA topoisomerases display similar, though less extreme, topologically-dependent differences in activity3. For example, human topoisomerase IIα preferentially relaxes positively supercoiled DNA, whereas the other human isoform, topoisomerase IIβ, relaxes positively and negatively supercoiled DNA with equal efficiency. The differences in activity among these enzymes can largely be attributed to differences in the poorly conserved C-terminal domains (CTDs), which contain a highly positively charged DNA binding surface4. Deletion of the gyrase GyrA CTD or topoisomerase IV ParC CTD results in a type IIA topoisomerase lacking chiral preference in relaxation and which no longer introduces negative supercoils4; 5. Indeed, chiral discrimination by topoisomerase IIA enzymes appears to be entirely dictated by the CTD domain. The chirality-dependent activity of chimeras between the human topoisomerase II α and β core enzymes with the alternative CTDs is almost entirely dictated by the CTDs rather than the core enzyme6. In addition, subtle mutations in a gyrase GyrA CTD result in an enzyme that exhibits topoisomerase IV-like chirality-dependent strand passage activity7. Whereas the domain responsible for chiral discrimination has been identified, the mechanistic basis for chiral discrimination by type IIA topoisomerases remains under debate. Proposed mechanisms include discrimination based on DNA crossing geometry and chirality-dependent differences in processivity4; 8; 9; 10; 11. Crystal structures of bacterial GyrA CTDs and ParC CTDs have not provided a great deal of additional insight. The GyrA and ParC CTDs are multi-bladed structures that contain a highly positively charged region that binds DNA 12. The similarity of the five blades found in E. coli ParC CTD suggests that it may simply provide an extended high-affinity DNA binding surface4; 12. Thus, despite determining the structure of the domain responsible for chiral discrimination by topoisomerase IV, the relationship between the structure and mechanism was not immediately clear. Figure 1 Topoisomerase IIA strand passage reaction. Topoisomerase binds gate segment DNA (blue) followed by binding of transfer segment DNA (yellow) and ATP that closes the N-gate (light blue). The gate segment is cleaved and the C-gate (light green) opens allowing ... In this issue, Vos, Lee, and Berger present the results of a systematic dissection of the role of key residues in each of the five blades of the topoisomerase IV Par-C CTD. By measuring the chirality-dependent relaxation and unlinking activity of point mutations of highly conserved basic residues in each blade, complemented by binding and bending assays of the intact enzyme as well as the isolated CTD, the authors made a number of striking discoveries. Rather than being a monolithic DNA binding element, each blade in the CTD appears to affect different aspects of topoisomerase IV activity in dramatically different manners. These results provide an important connection between the structure of the CTD and chirality-dependent modulation of strand passage activity. Remarkably, interactions between the DNA and the Par-C CTD appear to both stimulate and inhibit specific topology and substrate-dependent activities of topoisomerase IV. Perhaps the most interesting finding is that residues in blade 1, which is proximal to the N-terminal portion of the ParC domain, contribute to bending of the G-segment DNA bound by the core enzyme. Disruption of this residue leads to a severe decrease in activity across all measurements, consistent with the growing body of evidence establishing the importance of DNA bending for topoisomerase IIA activity13; 14. However, this is the first evidence that the ParC CTD participates in G-segment DNA interactions and it explains the dramatic decrease in activity observed in the topoisomerase IV ParC CTD deletion mutant4. The remaining blades modulate the activity of topoisomerase IV in distinct DNA substrate-dependent manners. Conserved basic residues in blades 2–4 modulate the activity, rate, and processivity of positive supercoil relaxation. Mutations of these residues decrease the overall activity of topoisomerase IV in relaxing positive supercoils. Paradoxically, these mutations slightly increase the relaxation rate while decreasing the processivity of positive supercoil relaxation. Mutations in blade 5 have little overall effect on the relaxation of positive supercoils. Remarkably, blade 5 appears to specifically inhibit negative supercoil relaxation as both the overall activity and rate increase when interactions between this blade and the DNA are disrupted. Negative supercoil relaxation appears to be governed by blades 2 and 3 as mutations in these blades decrease the overall activity, processivity, and rate of topoisomerase IV in relaxing negative supercoils. Mutations in blade 4 have virtually no effect on negative supercoil relaxation. Similar to negative supercoil relaxation, decatenation appears to be inhibited through the interaction of DNA with blade 5, but only blade 3 appears to be important for decatenation. Mutations in blades 2 and 4 have essentially no effect on decatenation activity or rate. The final surprising discovery by Vos and co-workers is that the effects of disrupting DNA binding at each blade did not uniformly decrease the overall binding affinity of the isolated CTD. Blades 1 and 2 appear to have little effect on DNA binding, whereas blades 3, 4, and 5 appear to play important roles in DNA binding, with mutations in blade 4 showing the strongest effect.

Fluorescent nanodiamonds (FNDs) emit in the near infrared and do not photo-bleach or photoblink. These properties make FNDs better suited for numerous imaging applications in comparison to commonly used fluorescence agents such as organic dyes and quantum dots. However, nanodiamonds do not form stable suspensions in aqueous buffer, are prone to aggregation, and are difficult to functionalize. Here, we present a method to encapsulate nanodiamonds with silica using an innovative liposome-based encapsulation process that renders the particle surface biocompatible, stable, and readily functionalized through routine linking chemistries. Furthermore, the method selects for a desired particle size and produces a monodisperse agent. We attached biotin to the silica-coated FNDs and tracked the three-dimensional motion of a biotinylated FND tethered by a single DNA molecule with high spatial and temporal resolution.

Summary Background Collagen, the most abundant human protein, is the principal component of the extracellular matrix and plays important roles in maintaining tissue and organ integrity. Highly resistant to proteolysis, fibrillar collagen is degraded by specific matrix metalloproteases (MMPs). Degradation of fibrillar collagen underlies processes including tissue remodeling, wound healing, and cancer metastasis. However, the mechanism of native collagen fibril degradation remains poorly understood. Results Here we present the results of high-resolution tracking of individual MMPs degrading type I collagen fibrils. MMP1 exhibits cleavage-dependent biased and hindered diffusion but spends 90% ± 3% of the time in one of at least two distinct pause states. One class of exponentially distributed pauses (class I pauses) occurs randomly along the fibril, whereas a second class of pauses (class II pauses) exhibits multistep escape kinetics and occurs periodically at intervals of 1.3 ± 0.2 μm and 1.5 ± 0.2 μm along the fibril. After these class II pauses, MMP1 moved faster and farther in one direction along the fibril, indicative of biased motion associated with cleavage. Simulations indicate that 5% ± 2% of the class II pauses result in the initiation of processive collagen degradation, which continues for bursts of 15 ± 4 consecutive cleavage events. Conclusions These findings provide a mechanistic paradigm for type I collagen degradation by MMP1 and establish a general approach to investigate MMP-fibrillar collagen interactions. More generally, this work demonstrates the fundamental role of enzyme-substrate interactions including binding and motion in determining the activity of an enzyme on an extended substrate.

Mitochondria and nuclei contain their own DNA and thus require topoisomerases for DNA metabolism. In vertebrates, the nuclear and mitochondrial topoisomerases IB are encoded by different genes. Although nuclear (nTop1) and mitochondrial (Top1mt) topoisomerases IB share a high degree of sequence similarity in their core domains, they are not transferable between the two organelles. To understand the mechanisms underlying this functional difference, we investigated the DNA relaxation activities of nTop1 and Top1mt using an in-vitro single molecule assay, and compared them to a N-terminal deletion mutant of nTop1 (Top68). Top68 provides insight into the functional significance of the N-terminal domain, which is largely missing in Top1mt. We characterized the topoisomerase catalytic steps by measuring uncoiling rate (turn/s), number of relaxed supercoils (uncoiling step-size), and time duration between nicking and resealing (religation time) as a function of the torque (twist) on the DNA. By adopting a periodic energy landscape model to characterize the differences among enzymes and using torque to probe uncoiling and religation, we find that Top1mt exhibits strong torque dependence as its uncoiling rate and average uncoiling step-size increase with increasing torque. On the other hand, the uncoiling rate and step-size of nTop1 are relatively insensitive to torque. The uncoiling rate and to a lesser extent the step-size of Top68 depends on torque, indicating a role of the N-terminal domain in DNA relaxation. These results demonstrate that protein domains distal to the DNA binding pocket and cleavage active site participate in the relaxation process including DNA rotation and religation.