Loop extrusion-mediated plasmid DNA cleavage by the bacterial SMC Wadjet complex.

Structural maintenance of chromosomes (SMC) protein complexes play pivotal roles in genome organization and maintenance across all domains of life. In prokaryotes, SMC family Wadjet complexes structurally resemble the widespread MukBEF genome-organizing complexes but serve a defensive role by inhibiting plasmid transformation. We previously showed that Wadjet specifically cleaves circular DNA; however, the molecular mechanism underlying DNA substrate recognition remains unclear. Here, we use in vitro single-molecule imaging to directly visualize DNA loop extrusion and plasmid cleavage by Wadjet. We find that Wadjet is a symmetric DNA loop extruder that simultaneously reels in DNA from both sides of a growing loop and that this activity requires a dimeric JetABC supercomplex containing two dimers of the JetC motor subunit. On surface-anchored plasmid DNAs, Wadjet extrudes the full length of a 44 kilobase pair plasmid, stalls, and then cleaves DNA. Our findings reveal the role of loop extrusion in the specific recognition and elimination of plasmids by Wadjet, and establish loop extrusion as an evolutionarily conserved mechanism among SMC complexes across kingdoms of life.

Loop-extruding Smc5/6 organizes transcription-induced positive DNA supercoils.

The structural maintenance of chromosomes (SMC) protein complexes-cohesin, condensin, and the Smc5/6 complex (Smc5/6)-are essential for chromosome function. At the molecular level, these complexes fold DNA by loop extrusion. Accordingly, cohesin creates chromosome loops in interphase, and condensin compacts mitotic chromosomes. However, the role of Smc5/6's recently discovered DNA loop extrusion activity is unknown. Here, we uncover that Smc5/6 associates with transcription-induced positively supercoiled DNA at cohesin-dependent loop boundaries on budding yeast (Saccharomyces cerevisiae) chromosomes. Mechanistically, single-molecule imaging reveals that dimers of Smc5/6 specifically recognize the tip of positively supercoiled DNA plectonemes and efficiently initiate loop extrusion to gather the supercoiled DNA into a large plectonemic loop. Finally, Hi-C analysis shows that Smc5/6 links chromosomal regions containing transcription-induced positive supercoiling in cis. Altogether, our findings indicate that Smc5/6 controls the three-dimensional organization of chromosomes by recognizing and initiating loop extrusion on positively supercoiled DNA.

Testing pseudotopological and nontopological models for SMC-driven DNA loop extrusion against roadblock-traversal experiments.

DNA loop extrusion by structural-maintenance-of-chromosome (SMC) complexes has emerged as a primary organizing principle for chromosomes. The mechanism by which SMC motor proteins extrude DNA loops is still unresolved and much debated. The ring-like structure of SMC complexes prompted multiple models where the extruded DNA is topologically or pseudotopologically entrapped within the ring during loop extrusion. However, recent experiments showed the passage of roadblocks much bigger than the SMC ring size, suggesting a nontopological mechanism. Recently, attempts were made to reconcile the observed passage of large roadblocks with a pseudotopological mechanism. Here we examine the predictions of these pseudotopological models and find that they are not consistent with new experimental data on SMC roadblock encounters. Particularly, these models predict the formation of two loops and that roadblocks will reside near the stem of the loop upon encounter-both in contrast to experimental observations. Overall, the experimental data reinforce the notion of a nontopological mechanism for extrusion of DNA.

The Smc5/6 complex is a DNA loop-extruding motor.

Structural maintenance of chromosomes (SMC) protein complexes are essential for the spatial organization of chromosomes1. Whereas cohesin and condensin organize chromosomes by extrusion of DNA loops, the molecular functions of the third eukaryotic SMC complex, Smc5/6, remain largely unknown2. Using single-molecule imaging, we show that Smc5/6 forms DNA loops by extrusion. Upon ATP hydrolysis, Smc5/6 reels DNA symmetrically into loops at a force-dependent rate of one kilobase pair per second. Smc5/6 extrudes loops in the form of dimers, whereas monomeric Smc5/6 unidirectionally translocates along DNA. We also find that the subunits Nse5 and Nse6 (Nse5/6) act as negative regulators of loop extrusion. Nse5/6 inhibits loop-extrusion initiation by hindering Smc5/6 dimerization but has no influence on ongoing loop extrusion. Our findings reveal functions of Smc5/6 at the molecular level and establish DNA loop extrusion as a conserved mechanism among eukaryotic SMC complexes.

SMC complexes can traverse physical roadblocks bigger than their ring size.

Ring-shaped structural maintenance of chromosomes (SMC) complexes like condensin and cohesin extrude loops of DNA. It remains, however, unclear how they can extrude DNA loops in chromatin that is bound with proteins. Here, we use in vitro single-molecule visualization to show that nucleosomes, RNA polymerase, and dCas9 pose virtually no barrier to loop extrusion by yeast condensin. We find that even DNA-bound nanoparticles as large as 200 nm, much bigger than the SMC ring size, also translocate into DNA loops during extrusion by condensin and cohesin. This even occurs for a single-chain version of cohesin in which the ring-forming subunits are covalently linked and cannot open to entrap DNA. The data show that SMC-driven loop extrusion has surprisingly little difficulty in accommodating large roadblocks into the loop. The findings also show that the extruded DNA does not pass through the SMC ring (pseudo)topologically, hence pointing to a nontopological mechanism for DNA loop extrusion.

Condensin-driven loop extrusion on supercoiled DNA.

Condensin, a structural maintenance of chromosomes (SMC) complex, has been shown to be a molecular motor protein that organizes chromosomes by extruding loops of DNA. In cells, such loop extrusion is challenged by many potential conflicts, for example, the torsional stresses that are generated by other DNA-processing enzymes. It has so far remained unclear how DNA supercoiling affects loop extrusion. Here, we use time-lapse single-molecule imaging to study condensin-driven DNA loop extrusion on supercoiled DNA. We find that condensin binding and DNA looping are stimulated by positively supercoiled DNA, and condensin preferentially binds near the tips of supercoiled plectonemes. Upon loop extrusion, condensin collects nearby plectonemes into a single supercoiled loop that is highly stable. Atomic force microscopy imaging shows that condensin generates supercoils in the presence of ATP. Our findings provide insight into the topology-regulated loading and formation of supercoiled loops by SMC complexes and clarify the interplay of loop extrusion and supercoiling.

ParB proteins can bypass DNA-bound roadblocks via dimer-dimer recruitment.

The ParABS system is essential for prokaryotic chromosome segregation. After loading at parS on the genome, ParB (partition protein B) proteins rapidly redistribute to distances of ~15 kilobases from the loading site. It has remained puzzling how this large-distance spreading can occur along DNA loaded with hundreds of proteins. Using in vitro single-molecule fluorescence imaging, we show that ParB from Bacillus subtilis can load onto DNA distantly of parS, as loaded ParB molecules themselves are found to be able to recruit additional ParB proteins from bulk. Notably, this recruitment can occur in cis but also in trans, where, at low tensions within the DNA, newly recruited ParB can bypass roadblocks as it gets loaded to spatially proximal but genomically distant DNA regions. The data are supported by molecular dynamics simulations, which show that cooperative ParB-ParB recruitment can enhance spreading. ParS-independent recruitment explains how ParB can cover substantial genomic distance during chromosome segregation, which is vital for the bacterial cell cycle.

Single electron transfer events and dynamical heterogeneity in the small protein azurin from Pseudomonas aeruginosa.

Monitoring the fluorescence of single-dye-labeled azurin molecules, we observed the reaction of azurin with hexacyanoferrate under controlled redox potential yielding data on the timing of individual (forward and backward) electron transfer (ET) events. Change-point analysis of the time traces demonstrates significant fluctuations of ET rates and of mid-point potential E 0. These fluctuations are a signature of dynamical heterogeneity, here observed on a 14 kDa protein, the smallest to date. By correlating changes in forward and backward reaction rates we found that 6% of the observed change events could be explained by a change in midpoint potential, while for 25% a change of the donor-acceptor coupling could explain the data. The remaining 69% are driven by variations in complex association constants or structural changes that cause forward and back ET rates to vary independently. Thus, the observed spread in individual ET rates could be related in a unique way to variations in molecular parameters. The relevance for the understanding of metabolic processes is briefly discussed.

Single-molecule fluorescence enhancement of a near-infrared dye by gold nanorods using DNA transient binding.

Fluorescence enhancement by plasmonic nanostructures enables the optical detection of single molecules with weak fluorescence, extending the scope of molecular fluorescence imaging to new materials and systems. In this work, we study single-molecule fluorescence enhancement by individual gold nanorods exploiting a DNA-based transient binding technique. Single molecules are attached to short DNA oligomers that can reversibly hybridize to their complementary docking DNA strands immobilized on the surface of gold nanorods or the glass substrate next to gold nanorods. This method continuously refreshes the single molecule in the near field of the gold nanorod, and enables a study of fluorescence enhancement at a well-defined position, with long dwell time and without limitation by photobleaching. Docking strands attached to the glass substrate are found to be more photo-stable. We find over 3000-fold fluorescence enhancement of single molecules of IRDye800CW, a near-infrared dye with a low quantum yield of 7%. This strong enhancement, consistent with numerical simulations, arises from the combined effect of local field enhancement and the competition between radiative and nonradiative decay rate enhancements.

Quantifying fluorescence enhancement for slowly diffusing single molecules in plasmonic near fields.

Gold nanorods are extensively used for single-molecule fluorescence enhancement as they are easy to synthesize, bio-compatible, and provide high light confinement at their nanometer-sized tips. The current way to estimate fluorescence enhancement relies on binned time traces or on fluorescence correlation spectroscopy. We report on novel ways to extract the enhancement factor in a single-molecule enhancement experiment, avoiding the arbitrary selection of one or a few high-intensity burst(s). These new estimates for the enhancement factor make use of the whole distribution of intensity bursts or of the interphoton delay distribution, which avoids the arbitrary binning of the fluorescence intensity time traces. We present experimental results on the bi-dimensional case, experimentally achieved using a lipid bilayer to support the diffusion of fluorophores. We support our findings with histograms of fluorescence bursts and with an analytical derivation of the interphoton delay distribution of (nearly) immobilized emitters from the fluorescence intensity profile.

Gold Nanorod Enhanced Fluorescence Enables Single-Molecule Electrochemistry of Methylene Blue.

Redox reactions are central to energy conversion and life metabolism. Herein we present electrochemical measurements with fluorescent readout of the redox-sensitive dye Methylene Blue (MB), at the single-molecule (SM) level. To overcome the low fluorescence quantum yield of MB we enhanced fluorescence by using individual gold nanorods to achieve the required sensitivity. By measuring the same molecule at different electrochemical potentials we determined the mid-point potential of each single molecule through its redox-induced fluorescence blinking dynamics.

Quantum work relations under trial Hamiltonians.

The universal quantum work relation connects a functional of an arbitrary observable averaged over the forward process to the free-energy difference and another functional averaged over the time-reversed process. Here, we ask the question if the system is driven out of equilibrium by a different Hamiltonian rather than the original one during the forward process and similarly during the reversed process then how accurate is the quantum work relation. We present an inequality that must be satisfied when the system is driven out by such a trial Hamiltonian. This also answers the issue of accuracy of the Jarzynski relation with a trial Hamiltonian. We have shown that the correction term can be expressed as the averages of the difference operator between the accurate and trial Hamiltonians. This leads to a generalized version of the Bogoliubov inequality for the free-energy differences.

Synthesis, structure, redox, NLO and DNA interaction aspects of [((L'-'")2RuII)3(mu3-L)]3+ and [(L')2RuII(NC5H4S-)]+[L3- = 1,3,5-triazine-2,4,6-trithiolato, L'-'" = arylazopyridine].

The trinuclear complexes [(L'-'")2RuII)3(mu3-L)](ClO4)3, [1] (ClO4)3-[3](ClO4)3 (L = trianionic form of 1,3,5-triazine-2,4,6-trithiol; N(p)C(5)H(4)N=N(a)-C6H4(R), R = H (L'), m-Me (L"), p-Me (L'")) and the analogous mononuclear complex [(L')2RuII(NC5H4S-)]ClO4 [4] ClO4 were synthesized. Crystal structures of [1](ClO4)3 and [4]ClO4 were determined. [1](3+)-[3](3+) exhibit three successive oxidative couples corresponding to Ru(II)Ru(II)Ru(III)<==>Ru(II)Ru(II)Ru(II); Ru(II)Ru(III)Ru(III)<==>Ru(II)Ru(II)Ru(III); Ru(III)Ru(III)Ru(III)<==>Ru(II)Ru(III)Ru(III) where the mixed valent states are moderately coupled. The complexes display multiple reductions associated with the azo functions of the ancillary ligands (L'-'"). The energy of the Ru(II)-based lowest energy MLCT transitions (533-558 nm) involving the pi* level of azoimine chromophore of L'-'" varies depending on the nuclearity as well as substituents in the ligand framework and follows the order: [1](3+) > [2](3+) > [3](3+) > [4](+). The complexes exhibit reasonably high third-order non-linear optical properties with gamma= (0.90-2.45) [times] 10(-29) esu. The interactions of the trinuclear complexes [((L')2RuII)3(mu3-L)]3+[1]3+, [((bpy)2RuII)3((mu3-L)]3+[5]3+ and [((phen)2RuII)3((mu3)-L)]3+[6]3+(bpy = 2,2'-bipyridine and phen = 1,10-phenanthroline) with the circular and linear forms of p-Bluescript DNA show reduced ethidium bromide fluorescence on gel electrophoresis.