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The discovery1,2and the pioneering applications3of CRISPR-Cas effector complexes have provided powerful gene-editing tools. The effector complexes are guided to the targeted genomic locus by the complementarity of their CRISPR RNA (crRNA)4,5. Recognition of double-stranded DNA targets proceeds via DNA unwinding and base-pairing between crRNA and the DNA target strand resulting in the formation of an R-loop structure5,6. Full R-loop formation is the prerequisite for the subsequent DNA cleavage. While the CRISPR-Cas technology is easy to use, efficient and highly versatile, therapeutic applications are hampered by the off-target effects due to the recognition of unintended sequences with multiple mismatches7. This process is still poorly understood on a mechanistic level8,9. Particularly, the lack of insight into the energetics and dynamics of the R-loop formation hinders a direct modelling of the R-loop formation for off-target prediction.Here we set up ultrafast DNA unwinding experiments based on plasmonic DNA nanorotors to follow the R-loop formation by the Cascade effector complex in real time, close to base pair resolution. We directly resolve a weak global downhill bias of the energy landscape of the forming R-loop followed by a steep uphill bias for the final base pairs. We furthermore show a modulation of the landscape by base flips and mismatches. These data provide that Cascade-mediated R-loop formation occurs on short time scales in single base pair steps of sub-millisecond duration, but on longer time scales in six–base pair intermediate steps in agreement with the structural periodicity of the crRNA-DNA hybrid. We expect that the knowledge about the energy landscapes of R-loop formation of CRISPR-Cas effector complexes will pave the way for a detailed understanding and prediction of off-target recognition10.

Cleavage of bacteriophage DNA by the Type III Restriction-Modification enzymes requires long-range interaction between DNA sites. This is facilitated by 1-dimensional diffusion (“DNA sliding”) initiated by ATP hydrolysis catalysed 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) for ATP hydrolysis. Our data shows that the helicase-like domain plays multiple roles. Firstly, stabilising initial DNA-interactions alongside the methyltransferase subunits. Secondly, causing environmental changes in the flipped adenine base following hydrolysis of the first ATP. And thirdly, remodelling nucleoprotein interactions via constrained translocation of a ~5-20 bp dsDNA loop. Initiation of DNA sliding requires 10-15 bp DNA downstream of the motor, corresponding to the site of nuclease domain binding. Our data unifies previous contradictory communication models for Type III enzymes.

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 first have to interrogate the entire genome for the presence of a matching sequence. Matching is achieved by base-pairing between the crRNA of the complexes and the DNA target strand such that an R-loop is formed. R-loop formation starts at a specific PAM motif and progresses reversibly in single base-pair steps until mismatches stop further progression or until the full target is recognized and destroyed. The reversible nature of this process entails that even a fully matching target should only become recognized with a low probability per target encounter. The details of this process, which directly affect the effectiveness of the target search, remain unresolved.Here we dissect the target search 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 low target recognition probabilities and show that they increase with increasing negative supercoiling. Furthermore, we demonstrate that Cascade uses a combination of three-dimensional and limited one-dimensional diffusion along the DNA contour for its target search. The latter allows for rapidly scanning the PAM sequences in a given region and, importantly, significantly increasing the overall efficiency of the target search by repeatedly revisiting the sites. Overall 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.

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

Here we demonstrate a strategy to rationally program a delayed onset of toehold-mediated DNA strand-displacement reactions. 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 the strand displacement reaction. The kinetics of the blocker enzymatic degradation thus controls the time at which the strand displacement reaction 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 the strand displacement reaction. 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 strand displacement reactions using RNase H and the two DNA repair enzymes Fpg and UDG and corresponding blockers. The achieved temporal delay can be programmed with high flexibility without undesired leak and can be conveniently predicted using kinetic modeling. Finally, we show that the delayed strand displacement reactions can be coupled to downstream processes and used to control the onset of ligand release from a DNA nanodevice as well as protein inhibition by a DNA aptamer.

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.

Nachdenken hilft! Auch beim Gespräch mit der nachfolgenden Generation über die Bedeutung der Vergangenheit und die Einordung des Erlebten. Ralf Seidel war über 20 Jahre Ärztlicher Direktor der Landesklinik Mönchengladbach. Bei Beginn seiner Arbeit als Psychiater hatte die Sachverständigenkommission für die Psychiatrie-Enquete gerade ihren ersten Zwischenbericht an den Bundestag übergeben. Ralf Seidel hat die Psychiatriereform in Deutschland nicht nur miterlebt, sondern auch mitgeprägt. Seine Interessen gingen dabei immer über sein Fach hinaus. Ralf Seidel hat nicht nur Medizin studiert, sondern auch Philosophie; er ist der Kunst sehr verbunden und weit gereist. Ein ungewöhnliches Arztleben zwischen Aufbruch, Reform und Reflexion.

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., Science Advances, 8 (5), ISSN:2375-2548

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.

The conserved Mre11-Rad50 (MR) complex is crucial for the detection, signaling, end tethering and processing of DNA double-strand breaks (DSBs). While it was known for decades that MR foci formation at DSBs 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 MR oligomerizes via a conserved Rad50 beta-sheet to higher-order assemblies, which bind DNA with positive cooperativity. We designed Rad50 point mutants with enhanced or disrupted MR oligomerization, and demonstrate that MR oligomerization facilitates foci formation, DNA damage signaling and repair in vivo. MR oligomerization does not affect its exonuclease activity but drives endonucleolytic cleavage at multiple sites on the 5'-terminated DNA strand near DSBs. Interestingly, mutations in the human Rad50 beta-sheet are linked to hereditary cancer predisposition and our findings might provide new insights into their potential role in chemoresistance.

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.

CRISPR-Cas9 is a ribonucleoprotein complex that sequence-specifically binds and cleaves double-stranded DNA. Wildtype Cas9 as well as 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 S. 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 non-target strand cleavage, supercoils are immediately but slowly released by swiveling of the non-target strand around the DNA with friction. Consequently, Cas9 and its non-target strand nicking mutant stay stably bound to the DNA for many hours even at elevated torsional stress. After 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 compared to the pre-cleavage state. This suggests that Cas9 has evolved to remain tightly bound to its cut target.

DNA intercalators bind nucleic acids by stacking between adjacent basepairs. This causes a considerable elongation of the DNA backbone as well as untwisting of the double helix. In the past few years, single-molecule mechanical experiments have become a common tool to characterize these deformations and to quantify important parameters of the intercalation process. Parameter extraction typically relies on the neighbor-exclusion model, in which a bound intercalator prevents intercalation into adjacent sites. Here, we challenge the neighbor-exclusion model by carefully quantifying and modeling the force-extension and twisting behavior of single ethidium-complexed DNA molecules. We show that only an anticooperative ethidium binding that allows for a disfavored but nonetheless possible intercalation into nearest-neighbor sites can consistently describe the mechanical behavior of intercalator-bound DNA. At high ethidium concentrations and elevated mechanical stress, this causes an almost complete occupation of nearest-neighbor sites and almost a doubling of the DNA contour length. We furthermore show that intercalation into nearest-neighbor sites needs to be considered when estimating intercalator parameters from zero-stress elongation and twisting data. We think that the proposed anticooperative binding mechanism may also be applicable to other intercalating molecules.

Recently introduced DNA nanomolds allow the shape-controlled growth of metallic nanoparticles. Here we demonstrate that this approach can be used to fabricate longer linear metal nanostructures of controlled lengths and patterns. To this end, we establish a set of different interfaces that enable mold interactions with high affinity and specificity. These interfaces enable and control the modular assembly of mold monomers into larger mold superstructure with programmable dimension in which each mold monomer remains uniquely addressable. Preloading the molds with nanoparticle seeds subsequently allows the growth of linear gold nanostructures whose lengths are controlled by the DNA structure. Exploiting the addressability of individual mold monomers furthermore allows achievement of site-specific metallization, that is, to create defined metal patterns. We think that the introduced approach provides a useful basis to fabricate nanomaterials with complex shapes and material composition in a fully programmable and modular fashion.

Im Mai 1992 hielt der 88-jahrige Hans Jonas im Munchner Prinzregententheater einen Vortrag mit dem Titel: »Philosophie. Ruckschau und Vorschau am Ende des Jahrhunderts«. So konnte Jonas noch einmal, ein knappes Jahr vor seinem Tod, so etwas wie die Summe seines philosophischen – und im weiteren Sinn auch politischen – Denkens entfalten. Weitere Vortragende dieser, von der mit Hans und Lore Jonas befreundeten Literaturwissenschaftlerin und Philosophin Rachel Salamander ins Leben gerufenen Veranstaltungsreihe waren u. a.: Joseph Brodsky, Henry Kissinger, Teddy Kollek, Derek Walcott, Hans-Georg Gadamer und Carl-Friedrich von Weizsacker. Im Folgenden werden die wesentlichen Inhalte des Vortrags von Jonas wiedergegeben.

To repair a DNA double-strand break by homologous recombination, 5'-terminated DNA strands must first be resected to reveal 3'-overhangs. This process is initiated by a short-range resection catalyzed by MRE11-RAD50-NBS1 (MRN) stimulated by CtIP, which is followed by a long-range step involving EXO1 or DNA2 nuclease. DNA2 is a bifunctional enzyme that contains both single-stranded DNA (ssDNA)-specific nuclease and motor activities. Upon DNA unwinding by Bloom (BLM) or Werner (WRN) helicase, RPA directs the DNA2 nuclease to degrade the 5'-strand. RPA bound to ssDNA also represents a barrier, explaining the need for the motor activity of DNA2 to displace RPA prior to resection. Using ensemble and single-molecule biochemistry, we show that CtIP also dramatically stimulates the adenosine 5'-triphosphate (ATP) hydrolysis-driven motor activity of DNA2 involved in the long-range resection step. This activation in turn strongly promotes the degradation of RPA-coated ssDNA by DNA2. Accordingly, the stimulatory effect of CtIP is only observed with wild-type DNA2, but not the helicase-deficient variant. Similarly to the function of CtIP to promote MRN, also the DNA2 stimulatory effect is facilitated by CtIP phosphorylation. The domain of CtIP required to promote DNA2 is located in the central region lacking in lower eukaryotes and is fully separable from domains involved in the stimulation of MRN. These results establish how CtIP couples both MRE11-dependent short-range and DNA2-dependent long-range resection and define the involvement of the motor activity of DNA2 in this process. Our data might help explain the less severe resection defects of MRE11 nuclease-deficient cells compared to those lacking CtIP.

We introduce a new concept for the solution-based fabrication of conductive gold nanowires using DNA templates. To this end, we employ DNA nanomolds, inside which electroless gold deposition is initiated by site-specific attached seeds. Using programmable interfaces, individual molds self-assemble into micrometer-long mold superstructures. During subsequent internal gold deposition, the mold walls constrain the metal growth, such that highly homogeneous nanowires with 20-30 nm diameters are obtained. Wire contacting using electron-beam lithography and electrical conductance characterization at temperatures between 4.2 K and room temperature demonstrate that metallic conducting wires were produced, although for part of the wires, the conductance is limited by boundaries between gold grains. Using different mold designs, our synthesis scheme will, in the future, allow the fabrication of complex metal structures with programmable shapes.

DNA double-strand break repair by homologous recombination employs long-range resection of the 5' DNA ends at the break points. In Saccharomyces cerevisiae, this process can be performed by the RecQ helicase Sgs1 and the helicase-nuclease Dna2. Though functional interplay between them has been shown, it remains unclear whether and how these proteins cooperate on the molecular level. Here, we resolved the dynamics of DNA unwinding by Sgs1 at the single-molecule level and investigated Sgs1 regulation by Dna2, the single-stranded DNA-binding protein RPA, and the Top3-Rmi1 complex. We found that Dna2 modulates the velocity of Sgs1, indicating that during end resection both proteins form a functional complex and couple their activities. Sgs1 drives DNA unwinding and feeds single-stranded DNA to Dna2 for degradation. RPA was found to regulate the processivity and the affinity of Sgs1 to the DNA fork, while Top3-Rmi1 modulated the velocity of Sgs1. We hypothesize that the differential regulation of Sgs1 activity by its protein partners is important to support diverse cellular functions of Sgs1 during the maintenance of genome stability.

DNA nanostructures provide a powerful platform for the programmable assembly of nanomaterials. Here, this approach is extended to semiconductor nanorods that possess interesting electrical properties and could be utilized for the bottom-up fabrication of nanoelectronic building blocks. The assembly scheme is based on an efficient DNA functionalization of the nanorods. A complete coverage of the rod surface with DNA ensures a high colloidal stability while maintaining the rod size and shape. It furthermore supports the assembly of the nanorods at defined docking positions of a DNA origami platform with binding efficiencies of up to 90 % as well as the formation of nanorod dimers with defined relative orientations. By incorporating orthogonal binding sites for gold nanoparticles, defined metal-semiconductor heterostructures can be fabricated. Subsequent application of a seeded growth procedure onto the gold nanoparticles (AuNPs) allows for to establish a direct metal-semiconductor interface as a crucial basis for the integration of semiconductors in self-assembled nanoelectronic devices.

DNA double-strand break repair by homologous recombination employs long-range resection of the 5’ DNA ends at the break points. In Saccharomyces cerevisiae, this process can be performed by the RecQ helicase Sgs1 and the helicase-nuclease Dna2. Though functional interplay has been shown, it remains unclear whether and how the proteins cooperate on the molecular level. Here, we resolved the dynamics of DNA unwinding by Sgs1 at the single molecule level and investigated its regulation by Dna2, the single-stranded DNA binding protein RPA and the Top3-Rmi1 complex. We found that Dna2 modulates the velocity of Sgs1, indicating that during end resection the proteins form a physical complex and couple their activities. Sgs1 unwinds DNA and feeds single-stranded DNA to Dna2 for degradation. RPA is found to regulate the processivity and the affinity of Sgs1 to the DNA fork, while Top3-Rmi1 modulated the velocity of Sgs1. We think that the differential regulation of the Sgs1 activity by its protein partners is important to allow diverse cellular functions of Sgs1 during the maintenance of genome stability.

Summary The multi-subunit type I CRISPR-Cas surveillance complex Cascade uses its crRNA to recognize dsDNA targets. Recognition involves DNA unwinding and base-pairing between the crRNA spacer region and a complementary DNA strand, resulting in formation of an R-loop structure. The modular Cascade architecture allows assembly of complexes containing crRNAs with altered spacer lengths that promise increased target specificity in emerging biotechnological applications. Here we produce type I-E Cascade complexes containing crRNAs with up to 57-nt-long spacers. We show that these complexes form R-loops corresponding to the designed target length, even for the longest spacers tested. Furthermore, the complexes can bind their targets with much higher affinity compared with the wild-type form. However, target recognition and the subsequent Cas3-mediated DNA cleavage do not require extended R-loops but already occur for wild-type-sized R-loops. These findings set important limits for specificity improvements of type I CRISPR-Cas systems., Graphical Abstract, Highlights • Cascades with elongated crRNA spacers support the formation of up to 57-bp-long R-loops • Target recognition and DNA degradation require only wild-type-sized, ∼30-bp-long R-loops • Complexes with elongated crRNAs bind targets with significantly increased affinity, Songailiene et al. show that engineered versions of Cascade with elongated crRNAs can base-pair with target DNA over 57 bp. However, target recognition only requires wild-type-sized base-pairing of ∼30-bp length. These findings define constraints for improving the specificity of these complexes in biotechnological applications.

The Bloom’s helicase ortholog, Sgs1, orchestrates the formation and disengagement of DNA joint molecules to enable controlled crossing-over during meiotic and mitotic DNA repair. Whether its enzymatic activity is temporally regulated to implement formation of noncrossovers prior to the activation of crossover-nucleases is unknown. Here, we show that akin to the Mus81-Mms4, Yen1 and MutLγ-Exo1 nucleases, Sgs1 helicase function is under tight cell cycle control through the actions of CDK and Polo kinase Cdc5. Notably, however, whereas CDK and Cdc5 unleash nuclease function during M-phase, they collaboratively delimit Sgs1 activation to S-phase. Mechanistically, CDK-mediated phosphorylation enhances the velocity and processivity of Sgs1, which stimulates DNA unwinding in vitro and joint molecule processing in vivo. Subsequent hyper-phosphorylation by Cdc5 appears to reduce the activity of Sgs1, while activating Mus81-Mms4 and MutLγ-Exo1. These findings suggest a general mechanism that drives orderly formation of noncrossover and crossover recombinants in meiotic and mitotic cells.