Mechanism of DNA unwinding by MCM8-9 in complex with HROB.

HROB promotes the MCM8-9 helicase in DNA damage response. To understand how HROB activates MCM8-9, we defined their interaction interface. We showed that HROB makes important yet transient contacts with both MCM8 and MCM9, and binds the MCM8-9 heterodimer with the highest affinity. MCM8-9-HROB prefer branched DNA structures, and display low DNA unwinding processivity. MCM8-9 unwinds DNA as a hexamer that assembles from dimers on DNA in the presence of ATP. The hexamer involves two repeating protein-protein interfaces between the alternating MCM8 and MCM9 subunits. One of these interfaces is quite stable and forms an obligate heterodimer across which HROB binds. The other interface is labile and mediates hexamer assembly, independently of HROB. The ATPase site formed at the labile interface contributes disproportionally more to DNA unwinding than that at the stable interface. Here, we show that HROB promotes DNA unwinding downstream of MCM8-9 loading and ring formation on ssDNA.

Heavy Metal Stabilization of DNA Origami Nanostructures.

DNA origami is a powerful tool to fold 3-dimensional DNA structures with nanometer precision. Its usage, however, is limited as high ionic strength, temperatures below ∼60 °C, and pH values between 5 and 10 are required to ensure the structural integrity of DNA origami nanostructures. Here, we demonstrate a simple and effective method to stabilize DNA origami nanostructures against harsh buffer conditions using [PdCl4]2-. It provided the stabilization of different DNA origami nanostructures against mechanical compression, temperatures up to 100 °C, double-distilled water, and pH values between 4 and 12. Additionally, DNA origami superstructures and bound cargos are stabilized with yields of up to 98%. To demonstrate the general applicability of our approach, we employed our protocol with a Pd metallization procedure at elevated temperatures. In the future, we think that our method opens up new possibilities for applications of DNA origami nanostructures beyond their usual reaction conditions.

Retention of the RNA ends provides the molecular memory for maintaining the activation of the Csm complex.

The type III CRISPR-Cas effector complex Csm functions as a molecular Swiss army knife that provides multilevel defense against foreign nucleic acids. The coordinated action of three catalytic activities of the Csm complex enables simultaneous degradation of the invader's RNA transcripts, destruction of the template DNA and synthesis of signaling molecules (cyclic oligoadenylates cAn) that activate auxiliary proteins to reinforce CRISPR-Cas defense. Here, we employed single-molecule techniques to connect the kinetics of RNA binding, dissociation, and DNA hydrolysis by the Csm complex from Streptococcus thermophilus. Although single-stranded RNA is cleaved rapidly (within seconds), dual-color FCS experiments and single-molecule TIRF microscopy revealed that Csm remains bound to terminal RNA cleavage products with a half-life of over 1 hour while releasing the internal RNA fragments quickly. Using a continuous fluorescent DNA degradation assay, we observed that RNA-regulated single-stranded DNase activity decreases on a similar timescale. These findings suggest that after fast target RNA cleavage the terminal RNA cleavage products stay bound within the Csm complex, keeping the Cas10 subunit activated for DNA destruction. Additionally, we demonstrate that during Cas10 activation, the complex remains capable of RNA turnover, i.e. of ongoing degradation of target RNA.

Short-range translocation by a restriction enzyme motor triggers diffusion along DNA.

Cleavage of bacteriophage DNA by the Type III restriction-modification enzymes requires long-range interaction between DNA sites. This is facilitated by one-dimensional diffusion ('DNA sliding') initiated by ATP hydrolysis catalyzed by a superfamily 2 helicase-like ATPase. Here we combined ultrafast twist measurements based on plasmonic DNA origami nano-rotors with stopped-flow fluorescence and gel-based assays to examine the role(s) of ATP hydrolysis. Our data show that the helicase-like domain has multiple roles. First, this domain stabilizes initial DNA interactions alongside the methyltransferase subunits. Second, it causes environmental changes in the flipped adenine base following hydrolysis of the first ATP. Finally, it remodels nucleoprotein interactions via constrained translocation of a ∼ 5 to 22-bp double stranded DNA loop. Initiation of DNA sliding requires 8-15 bp of DNA downstream of the motor, corresponding to the site of nuclease domain binding. Our data unify previous contradictory communication models for Type III enzymes.

Innate programmable DNA binding by CRISPR-Cas12m effectors enable efficient base editing.

Cas9 and Cas12 nucleases of class 2 CRISPR-Cas systems provide immunity in prokaryotes through RNA-guided cleavage of foreign DNA. Here we characterize a set of compact CRISPR-Cas12m (subtype V-M) effector proteins and show that they provide protection against bacteriophages and plasmids through the targeted DNA binding rather than DNA cleavage. Biochemical assays suggest that Cas12m effectors can act as roadblocks inhibiting DNA transcription and/or replication, thereby triggering interference against invaders. Cryo-EM structure of Gordonia otitidis (Go) Cas12m ternary complex provided here reveals the structural mechanism of DNA binding ensuring interference. Harnessing GoCas12m innate ability to bind DNA target we fused it with adenine deaminase TadA-8e and showed an efficient A-to-G editing in Escherichia coli and human cells. Overall, this study expands our understanding of the functionally diverse Cas12 protein family, revealing DNA-binding dependent interference mechanism of Cas12m effectors that could be harnessed for engineering of compact base-editing tools.

Timed Pulses in DNA Strand Displacement Reactions.

Inspired by naturally occurring regulatory mechanisms that allow complex temporal pulse features with programmable delays, we demonstrate here a strategy to achieve temporally programmed pulse output signals in DNA-based strand displacement reactions (SDRs). To achieve this, we rationally designed input strands that, once bound to their target duplex, can be gradually degraded, resulting in a pulse output signal. We also designed blocker strands that suppress strand displacement and determine the time at which the pulse reaction is generated. We show that by controlling the degradation rate of blocker and input strands, we can finely control the delayed pulse output over a range of 10 h. We also prove that it is possible to orthogonally delay two different pulse reactions in the same solution by taking advantage of the specificity of the degradation reactions for the input and blocker strands. Finally, we show here two possible applications of such delayed pulse SDRs: the time-programmed pulse decoration of DNA nanostructures and the sequentially appearing and self-erasing formation of DNA-based patterns.

Mechanism of DNA unwinding by hexameric MCM8-9 in complex with HROB.

The human MCM8-9 helicase functions in concert with HROB in the context of homologous recombination, but its precise function is unknown. To gain insights into how HROB regulates MCM8-9, we first used molecular modeling and biochemistry to define their interaction interface. We show that HROB makes important contacts with both MCM8 and MCM9 subunits, which directly promotes its DNA-dependent ATPase and helicase activities. MCM8-9-HROB preferentially binds and unwinds branched DNA structures, and single-molecule experiments reveal a low DNA unwinding processivity. MCM8-9 unwinds DNA as a hexameric complex that assembles from dimers on DNA in the presence of ATP, which is prerequisite for its helicase function. The hexamer formation thus involves two repeating protein-protein interfaces forming between the alternating MCM8 and MCM9 subunits. One of these interfaces is rather stable and forms an obligate heterodimer, while the other interface is labile and mediates the assembly of the hexamer on DNA, independently of HROB. The ATPase site composed of the subunits forming the labile interface disproportionally contributes to DNA unwinding. HROB does not affect the MCM8-9 ring formation, but promotes DNA unwinding downstream by possibly coordinating ATP hydrolysis with structural transitions accompanying translocation of MCM8-9 on DNA.

Mechanism of DNA unwinding by hexameric MCM8-9 in complex with HROB.

The human MCM8-9 helicase functions in concert with HROB in the context of homologous recombination, but its precise function is unknown. To gain insights into how HROB regulates MCM8-9, we first used molecular modeling and biochemistry to define their interaction interface. We show that HROB makes important contacts with both MCM8 and MCM9 subunits, which directly promotes its DNA-dependent ATPase and helicase activities. MCM8-9-HROB preferentially binds and unwinds branched DNA structures, and single-molecule experiments reveal a low DNA unwinding processivity. MCM8-9 unwinds DNA as a hexameric complex that assembles from dimers on DNA in the presence of ATP, which is prerequisite for its helicase function. The hexamer formation thus involves two repeating protein-protein interfaces forming between the alternating MCM8 and MCM9 subunits. One of these interfaces is rather stable and forms an obligate heterodimer, while the other interface is labile and mediates the assembly of the hexamer on DNA, independently of HROB. The ATPase site composed of the subunits forming the labile interface disproportionally contributes to DNA unwinding. HROB does not affect the MCM8-9 ring formation, but promotes DNA unwinding downstream by possibly coordinating ATP hydrolysis with structural transitions accompanying translocation of MCM8-9 on DNA.

The energy landscape for R-loop formation by the CRISPR-Cas Cascade complex.

Clustered regularly interspaced short palindromic repeats (CRISPR) sequences and CRISPR-associated (Cas) genes comprise CIRSPR-Cas effector complexes, which have revolutionized gene editing with their ability to target specific genomic loci using CRISPR RNA (crRNA) complementarity. Recognition of double-stranded DNA targets proceeds via DNA unwinding and base pairing between crRNA and the DNA target strand, forming an R-loop structure. Full R-loop extension is a prerequisite for subsequent DNA cleavage. However, the recognition of unintended sequences with multiple mismatches has limited therapeutic applications and is still poorly understood on a mechanistic level. Here we set up ultrafast DNA unwinding experiments on the basis of plasmonic DNA origami nanorotors to study R-loop formation by the Cascade effector complex in real time, close to base-pair resolution. We resolve a weak global downhill bias of the forming R-loop, followed by a steep uphill bias for the final base pairs. We also show that the energy landscape is modulated by base flips and mismatches. These findings suggest that Cascade-mediated R-loop formation occurs on short timescales in submillisecond single base-pair steps, but on longer timescales in six base-pair intermediate steps, in agreement with the structural periodicity of the crRNA-DNA hybrid.

Dynamic interplay between target search and recognition for a Type I CRISPR-Cas system.

CRISPR-Cas effector complexes enable the defense against foreign nucleic acids and have recently been exploited as molecular tools for precise genome editing at a target locus. To bind and cleave their target, the CRISPR-Cas effectors have to interrogate the entire genome for the presence of a matching sequence. Here we dissect the target search and recognition process of the Type I CRISPR-Cas complex Cascade by simultaneously monitoring DNA binding and R-loop formation by the complex. We directly quantify the effect of DNA supercoiling on the target recognition probability and demonstrate that Cascade uses facilitated diffusion for its target search. We show that target search and target recognition are tightly linked and that DNA supercoiling and limited 1D diffusion need to be considered when understanding target recognition and target search by CRISPR-Cas enzymes and engineering more efficient and precise variants.

DNA Mold-Based Fabrication of Palladium Nanostructures.

DNA origami molds allow a shape-controlled growth of metallic nanoparticles. So far, this approach is limited to gold and silver. Here, the fabrication of linear palladium nanostructures with controlled lengths and patterns is demonstrated. To obtain nucleation centers for a seeded growth, a synthesis procedure of palladium nanoparticles (PdNPs) using Bis(p-sulfonatophenyl)phenylphosphine (BSPP) both as reductant and stabilizer is developed to establish an efficient functionalization protocol of the particles with single-stranded DNA. Attaching the functionalized particles to complementary DNA strands inside DNA mold cavities supports subsequently a highly specific seeded palladium deposition. This provides rod-like PdNPs with diameters of 20-35 nm of grainy morphology. Using an annealing procedure and a post-reduction step with hydrogen, homogeneous palladium nanostructures can be obtained. With the adaptation of the procedure to palladium the capabilities of the mold-based tool-box are expanded. In the future, this may allow a facile adaptation of the mold approach to less noble metals including magnetic materials such as Ni and Co.

A quantitative model for the dynamics of target recognition and off-target rejection by the CRISPR-Cas Cascade complex.

CRISPR-Cas effector complexes recognise nucleic acid targets by base pairing with their crRNA which enables easy re-programming of the target specificity in rapidly emerging genome engineering applications. However, undesired recognition of off-targets, that are only partially complementary to the crRNA, occurs frequently and represents a severe limitation of the technique. Off-targeting lacks comprehensive quantitative understanding and prediction. Here, we present a detailed analysis of the target recognition dynamics by the Cascade surveillance complex on a set of mismatched DNA targets using single-molecule supercoiling experiments. We demonstrate that the observed dynamics can be quantitatively modelled as a random walk over the length of the crRNA-DNA hybrid using a minimal set of parameters. The model accurately describes the recognition of targets with single and double mutations providing an important basis for quantitative off-target predictions. Importantly the model intrinsically accounts for observed bias regarding the position and the proximity between mutations and reveals that the seed length for the initiation of target recognition is controlled by DNA supercoiling rather than the Cascade structure.

Trapping of protein cargo molecules inside DNA origami nanocages.

The development of the DNA origami technique has directly inspired the idea of using three-dimensional DNA cages for the encapsulation and targeted delivery of drug or cargo molecules. The cages would be filled with molecules that would be released at a site of interest upon cage opening triggered by an external stimulus. Though different cage variants have been developed, efficient loading of DNA cages with freely-diffusing cargo molecules that are not attached to the DNA nanostructure and their efficient retention within the cages has not been presented. Here we address these challenges using DNA origami nanotubes formed by a double-layer of DNA helices that can be sealed with tight DNA lids at their ends. In a first step we attach DNA-conjugated cargo proteins to complementary target strands inside the DNA tubes. After tube sealing, the cargo molecules are released inside the cavity using toehold-mediated strand displacement by externally added invader strands. We show that DNA invaders are rapidly entering the cages through their DNA walls. Retention of ∼70 kDa protein cargo molecules inside the cages was, however, poor. Guided by coarse-grained simulations of the DNA cage dynamics, a tighter sealing of the DNA tubes was developed which greatly reduced the undesired escape of cargo proteins. These improved DNA nanocages allow for efficient encapsulation of medium-sized cargo molecules while remaining accessible to small molecules that can be used to trigger reactions, including a controlled release of the cargo via nanocage opening.

Orthogonal Enzyme-Driven Timers for DNA Strand Displacement Reactions.

Here, we demonstrate a strategy to rationally program a delayed onset of toehold-mediated DNA strand displacement reactions (SDRs). The approach is based on blocker strands that efficiently inhibit the strand displacement by binding to the toehold domain of the target DNA. Specific enzymatic degradation of the blocker strand subsequently enables SDR. The kinetics of the blocker enzymatic degradation thus controls the time at which the SDR starts. By varying the concentration of the blocker strand and the concentration of the enzyme, we show that we can finely tune and modulate the delayed onset of SDR. Additionally, we show that the strategy is versatile and can be orthogonally controlled by different enzymes each specifically targeting a different blocker strand. We designed and established three different delayed SDRs using RNase H and two DNA repair enzymes (formamidopyrimidine DNA glycosylase and uracil-DNA glycosylase) and corresponding blockers. The achieved temporal delay can be programed with high flexibility without undesired leak and can be conveniently predicted using kinetic modeling. Finally, we show three possible applications of the delayed SDRs to temporally control the ligand release from a DNA nanodevice, the inhibition of a target protein by a DNA aptamer, and the output signal generated by a DNA logic circuit.

Mre11-Rad50 oligomerization promotes DNA double-strand break repair.

The conserved Mre11-Rad50 complex is crucial for the detection, signaling, end tethering and processing of DNA double-strand breaks. While it is known that Mre11-Rad50 foci formation at DNA lesions accompanies repair, the underlying molecular assembly mechanisms and functional implications remained unclear. Combining pathway reconstitution in electron microscopy, biochemical assays and genetic studies, we show that S. cerevisiae Mre11-Rad50 with or without Xrs2 forms higher-order assemblies in solution and on DNA. Rad50 mediates such oligomerization, and mutations in a conserved Rad50 beta-sheet enhance or disrupt oligomerization. We demonstrate that Mre11-Rad50-Xrs2 oligomerization facilitates foci formation, DNA damage signaling, repair, and telomere maintenance in vivo. Mre11-Rad50 oligomerization does not affect its exonuclease activity but drives endonucleolytic cleavage at multiple sites on the 5'-DNA strand near double-strand breaks. Interestingly, mutations in the human RAD50 beta-sheet are linked to hereditary cancer predisposition and our findings might provide insights into their potential role in chemoresistance.

Dissipative Control over the Toehold-Mediated DNA Strand Displacement Reaction.

Here we show a general approach to achieve dissipative control over toehold-mediated strand-displacement, the most widely employed reaction in the field of DNA nanotechnology. The approach relies on rationally re-engineering the classic strand displacement reaction such that the high-energy invader strand (fuel) is converted into a low-energy waste product through an energy-dissipating reaction allowing the spontaneous return to the original state over time. We show that such dissipative control over the toehold-mediated strand displacement process is reversible (up to 10 cycles), highly controllable and enables unique temporal activation of DNA systems. We show here two possible applications of this strategy: the transient labelling of DNA structures and the additional temporal control of cascade reactions.

The CDK1-TOPBP1-PLK1 axis regulates the Bloom's syndrome helicase BLM to suppress crossover recombination in somatic cells.

Bloom's syndrome is caused by inactivation of the BLM helicase, which functions with TOP3A and RMI1-2 (BTR complex) to dissolve recombination intermediates and avoid somatic crossing-over. We show here that crossover avoidance by BTR further requires the activity of cyclin-dependent kinase-1 (CDK1), Polo-like kinase-1 (PLK1), and the DDR mediator protein TOPBP1, which act in the same pathway. Mechanistically, CDK1 phosphorylates BLM and TOPBP1 and promotes the interaction of both proteins with PLK1. This is amplified by the ability of TOPBP1 to facilitate phosphorylation of BLM at sites that stimulate both BLM-PLK1 and BLM-TOPBP1 binding, creating a positive feedback loop that drives rapid BLM phosphorylation at the G2-M transition. In vitro, BLM phosphorylation by CDK/PLK1/TOPBP1 stimulates the dissolution of topologically linked DNA intermediates by BLM-TOP3A. Thus, we propose that the CDK1-TOPBP1-PLK1 axis enhances BTR-mediated dissolution of recombination intermediates late in the cell cycle to suppress crossover recombination and curtail genomic instability.

Probing the stability of the SpCas9-DNA complex after cleavage.

CRISPR-Cas9 is a ribonucleoprotein complex that sequence-specifically binds and cleaves double-stranded DNA. Wildtype Cas9 and its nickase and cleavage-incompetent mutants have been used in various biological techniques due to their versatility and programmable specificity. Cas9 has been shown to bind very stably to DNA even after cleavage of the individual DNA strands, inhibiting further turnovers and considerably slowing down in-vivo repair processes. This poses an obstacle in genome editing applications. Here, we employed single-molecule magnetic tweezers to investigate the binding stability of different Streptococcus pyogenes Cas9 variants after cleavage by challenging them with supercoiling. We find that different release mechanisms occur depending on which DNA strand is cleaved. After initial target strand cleavage, supercoils are only removed after the collapse of the R-loop. We identified several states with different stabilities of the R-loop. Most importantly, we find that the post-cleavage state of Cas9 exhibits a higher stability than the pre-cleavage state. After non-target strand cleavage, supercoils are immediately but slowly released by swiveling of the non-target strand around Cas9 bound to the target strand. Consequently, Cas9 and its non-target strand nicking mutant stay stably bound to the DNA for many hours even at elevated torsional stress.

Distinct RPA domains promote recruitment and the helicase-nuclease activities of Dna2.

The Dna2 helicase-nuclease functions in concert with the replication protein A (RPA) in DNA double-strand break repair. Using ensemble and single-molecule biochemistry, coupled with structure modeling, we demonstrate that the stimulation of S. cerevisiae Dna2 by RPA is not a simple consequence of Dna2 recruitment to single-stranded DNA. The large RPA subunit Rfa1 alone can promote the Dna2 nuclease activity, and we identified mutations in a helix embedded in the N-terminal domain of Rfa1 that specifically disrupt this capacity. The same RPA mutant is instead fully functional to recruit Dna2 and promote its helicase activity. Furthermore, we found residues located on the outside of the central DNA-binding OB-fold domain Rfa1-A, which are required to promote the Dna2 motor activity. Our experiments thus unexpectedly demonstrate that different domains of Rfa1 regulate Dna2 recruitment, and its nuclease and helicase activities. Consequently, the identified separation-of-function RPA variants are compromised to stimulate Dna2 in the processing of DNA breaks. The results explain phenotypes of replication-proficient but radiation-sensitive RPA mutants and illustrate the unprecedented functional interplay of RPA and Dna2.

Chemical ligation of an entire DNA origami nanostructure.

Within the field of DNA nanotechnology, numerous methods were developed to produce complex two- and three-dimensional DNA nanostructures for many different emerging applications. These structures typically suffer from a low tolerance against non-optimal environmental conditions including elevated temperatures. Here, we apply a chemical ligation method to covalently seal the nicks between adjacent 5' phosphorylated and 3' amine-modified strands within the DNA nanostructures. Using a cost-effective enzymatic strand modification procedure, we are able to batch-modify all DNA strands even of large DNA objects, such as origami nanostructures. The covalent strand linkage increases the temperature stability of the structures by ∼10 K. Generally, our method also allows a 'surgical' introduction of covalent strand linkages at preselected positions. It can also be used to map the strand ligation into chains throughout the whole nanostructure and identify assembly defects. We expect that our method can be applied to a large variety of DNA nanostructures, in particular when full control over the introduced covalent linkages and the absence of side adducts and DNA damages are required.