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DNA double-strand breaks in DNA (DSBs) are common yet highly detrimental events in living organisms. To repair the damage, each cell requires a coordinated set of DNA damage response (DDR) proteins that can respond quickly, effectively, and precisely. Better understanding of these processes is therefore essential and would require an effective means of inducing targeted DSBs on demand, but previous methods are hampered by limited control over genomic location, timing, or lesion types. Tight spatiotemporal control of CRISPR-Cas9 activity has potential to overcome these limitations, which led to the development of two methods for rapid activation or deactivation of Cas9 using light. In this chapter, we discuss how control of Cas9 can advance DDR studies, describe protocols to control Cas9 activation and deactivation using this new technology, and finally outline three compatible readouts of DNA damage and the cellular response: DSB levels using droplet digital PCR, repair factor localization using ChIP-seq, and insertion-deletion (indel) repair outcomes using Sanger sequencing.

Summary The ability to deactivate CRISPR-Cas systems on demand would improve the safety and applicability of genome editing. Here, we detail a protocol using photocleavable guide RNAs (pcRNAs) to deactivate CRISPR-Cas9 inside cells. We verify that deactivation is both rapid and complete by checking for insertion-deletion (indel) mutations using Sanger sequencing. This protocol will be useful for researchers interested in using pcRNAs to improve genome editing specificity, characterize the timescales of genome editing, and study cellular DNA damage responses. For complete details on the use and execution of this protocol, please refer to Zou et al. (2021)., Graphical abstract, Highlights • Protocol using photocleavable guide RNAs to deactivate CRISPR-Cas9 inside cells • Deactivation is both rapid and complete by checking for insertion-deletion mutations • Useful for characterizing genome editing dynamics and DNA repair processes, The ability to deactivate CRISPR-Cas systems on demand would improve the safety and applicability of genome editing. Here, we detail a protocol using photocleavable guide RNAs (pcRNAs) to deactivate CRISPR-Cas9 inside cells. We verify that deactivation is both rapid and complete by checking for insertion-deletion (indel) mutations using Sanger sequencing. This protocol will be useful for researchers interested in using pcRNAs to improve genome editing specificity, characterize the timescales of genome editing, and study cellular DNA damage responses.

The sequence-specific hybridization of DNA facilitates its use as a building block for designer nanoscale structures and reaction networks that perform computations. However, the strong binding energy of Watson-Crick base pairing that underlies this specificity also causes the DNA dehybridization rate to depend sensitively on sequence length and temperature. This strong dependency imposes stringent constraints on the design of multi-step DNA reactions, because a small deviation from the optimal conditions slows down the process dramatically. Here we show how an ATP-dependent helicase, Rep-X, can drive certain dehybridization reactions in designed DNA reaction networks at rates independent of sequence length, thereby decoupling the rates of hybridization and dehybridization. To illustrate this principle, we show that Rep-X extends the range of conditions where the primer exchange reaction, which catalytically adds a domain provided by a hairpin template to a DNA substrate, proceeds rapidly: in the strong substrate-hairpin binding regime, Rep-X expedites the reaction almost one hundred-fold. Our results provide an example of how ATP consumption can drive specific dehybridization reactions in designed DNA reaction networks and how this consumption can be harnessed to expedite reactions beyond their equilibrium rates.

CRISPR-Cas9 has transformed biomedical research and medicine through convenient and targeted manipulation of DNA. Time- and spatially-resolved control over Cas9 activity through the recently developed very fast CRISPR (vfCRISPR) system have facilitated comprehensive studies of DNA damage and repair. Understanding the fundamental principles of Cas9 binding and cleavage behavior is essential before the widespread use of these systems and can be readily accomplished in vitro through both cleavage and electrophoretic mobility shift assays (EMSA). The protocol for in vitro cleavage consists of Cas9 with guide RNA (gRNA) ribonucleoprotein (RNP) formation, followed by incubation with target DNA. For EMSA, this reaction is directly loaded onto an agarose gel for visualization of the target DNA band that is shifted due to binding by the Cas9 RNP. To assay for cleavage, Proteinase K is added to degrade the RNP, allowing target DNA (cleaved and/or uncleaved) to migrate consistently with its molecular weight. Heating at 95°C rapidly inactivates the RNP on demand, allowing time-resolved measurements of Cas9 cleavage kinetics. This protocol facilitates the characterization of the light-activation mechanism of photocaged vfCRISPR gRNA.

Dynamin mediates fission of vesicles from the plasma membrane during endocytosis. Typically, dynamin is recruited from the cytosol to endocytic sites, requiring seconds to tens of seconds. However, ultrafast endocytosis in neurons internalizes vesicles as quickly as 50 ms during synaptic vesicle recycling. Here, we demonstrate that Dynamin 1 is pre-recruited to endocytic sites for ultrafast endocytosis. Specifically, Dynamin 1xA, a splice variant of Dynamin 1, interacts with Syndapin 1 to form molecular condensates on the plasma membrane. Single-particle tracking of Dynamin 1xA molecules confirms the liquid-like property of condensates in vivo. When Dynamin 1xA is mutated to disrupt its interaction with Syndapin 1, the condensates do not form, and consequently, ultrafast endocytosis slows down by 100-fold. Mechanistically, Syndapin 1 acts as an adaptor by binding the plasma membrane and stores Dynamin 1xA at endocytic sites. This cache bypasses the recruitment step and accelerates endocytosis at synapses.

Mechanical response to external stimuli is a conserved feature of many cell types. For example, neurotransmitters (e.g., histamine) trigger calcium signals that induce actomyosin-regulated contraction of airway smooth muscle (ASM); the resulting cell shortening causes airway narrowing, the excess of which can cause asthma. Despite intensive studies, however, it remains unclear how physical forces are propagated through focal adhesion (FA)-the major force-transmission machinery of the cell-during ASM shortening. We provide a nanomechanical platform to directly image single molecule forces during ASM cell shortening by repurposing DNA tension sensors. Surprisingly, cell shortening and FA disassembly that immediately precedes it occurred long after histamine-evoked increases in intracellular calcium levels ([Casup2+/sup]subi/sub). Our mathematical model that fully integrates cell edge protrusion and retraction with contractile forces acting on FA predicted that (1) stabilization of FA impedes cell shortening and (2) the disruption of FAs is preceded by their strengthening through actomyosin-activated molecular tension. We confirmed these predictions via real-time imaging and molecular force measurements. Together, our work highlights a key role of FA dynamics in regulating ASM contraction induced by an allergen with potential therapeutic implications.

Pif1 plays multiple roles in maintaining genome stability and preferentially unwinds forked dsDNA, but the mechanism by which Pif1 unwinds forked dsDNA remains elusive. Here we report the structure of Bacteroides sp Pif1 (BaPif1) in complex with a symmetrical double forked dsDNA. Two interacting BaPif1 molecules are bound to each fork of the partially unwound dsDNA, and interact with the 5′ arm and 3′ ss/dsDNA respectively. Each of the two BaPif1 molecules is an active helicase and their interaction may regulate their helicase activities. The binding of BaPif1 to the 5′ arm causes a sharp bend in the 5′ ss/dsDNA junction, consequently breaking the first base-pair. BaPif1 bound to the 3′ ss/dsDNA junction impacts duplex unwinding by stabilizing the unpaired first base-pair and engaging the second base-pair poised for breaking. Our results provide an unprecedented insight into how two BaPif1 coordinate with each other to unwind the forked dsDNA., Pif1 plays multiple roles in maintaining genome stability and preferentially unwinds forked dsDNA. Here the authors solve the structure of Bacteroides sp Pif1 (BaPif1) in complex with a symmetrical double forked dsDNA and provide unprecedented insights into forked dsDNA unwinding by BaPif1.

A human telomere ends in a single-stranded 3′ tail, composed of repeats of T2AG3. G-quadruplexes (GQs) formed from four consecutive repeats have been shown to possess high-structural and mechanical diversity. In principle, a GQ can form from any four repeats that are not necessarily consecutive. To understand the dynamics of GQs with positional multiplicity, we studied five and six repeats human telomeric sequence using a combination of single molecule FRET and optical tweezers. Our results suggest preferential formation of GQs at the 3′ end both in K+ and Na+ solutions, with minor populations of 5′-GQ or long-loop GQs. A vectorial folding assay which mimics the directional nature of telomere extension showed that the 3′ preference holds even when folding is allowed to begin from the 5′ side. In 100 mM K+, the unassociated T2AG3 segment has a streamlining effect in that one or two mechanically distinct species was observed at a single position instead of six or more observed without an unassociated repeat. We did not observe such streamlining effect in 100 mM Na+. Location of GQ and reduction in conformational diversity in the presence of extra repeats have implications in telomerase inhibition, T-loop formation and telomere end protection.

STED nanoscopy helped usher in resolution revolution of optical microscopy and has been widely used to study subcellular structures and dynamics. To achieve optical resolution beyond the diffractio...

Holliday junction (HJ) resolution by its resolving enzymes is essential for chromosome segregation and recombination-mediated DNA repair. HJs undergo two types of structural dynamics that determine the outcome of recombination: conformer exchange between two isoforms and branch migration. However, it is unknown how the preferred branch-point and conformer are achieved between enzyme binding and HJ resolution given the extensive binding interactions seen in static crystal structures. Single molecule fluorescence resonance energy transfer analysis of resolving-enzymes from bacteriophages (T7 endonuclease I), bacteria (RuvC), fungi (GEN1) and humans (hMus81-Eme1) showed that both types of HJ dynamics still occur after enzyme binding. These dimeric enzymes use their multivalent interactions to achieve this, going through a partially-dissociated intermediate in which the HJ undergoes nearly unencumbered dynamics. This evolutionarily conserved property of HJ resolving-enzymes provides previously unappreciated insight on how junction resolution, conformer exchange and branch migration may be coordinated.

Cells must respond specifically and dynamically to mechanical cues from the extracellular environment and dysregulation of extracellular force sensing leads to a variety of diseases. Therefore, it is important to deconvolve the many inputs that transduce mechanical signals and understand how these signals are interpreted and responded to. DNA and peptide-based molecular force sensors have been previously developed to measure forces applied through single membrane receptors including integrins and Notch receptors. The tension gauge tether (TGT) exploits the physical rupture force of double-stranded DNA to measure and modulate the force applied through single receptor-ligand bonds and can cover a wide range of tension (10-60 pN). By exploiting a fluorescent dye-quencher pair and collecting differential fluorescence signals over time, we characterized the quenched tension gauge tether (qTGT) system and developed an image analysis protocol to measure molecular tension in quasi-real time. We show that this differential qTGT analysis method can simultaneously measure multiple levels of integrin-mediated molecular tension over a wide time scale during the onset of adhesion and cell migration.

The circumsporozoite protein (CSP) forms a dense coat on the surface of the sporozoite, the infective stage of the malaria parasite. The central repeat region of CSP is a critical component of the only licensed malaria vaccine yet little is known about its structure or function. We found that sporozoite mutants with severely truncated or scrambled repeats have impaired motility due to altered adhesion site formation and dynamics, suggesting that the CSP repeats provide a cohesive environment in which adhesion sites can form. We hypothesized that biophysical properties of the repeats are important in this role and interrogated this using single-molecule fluorescence-force spectroscopy. We show that the repeats are a stiff, linear spring with elastic properties, dependent upon length and lost when the repeats are scrambled. These data are the first evidence that the CSP repeat region serves a functional role during infection and motility, likely mediated through its biophysical properties.SummaryNo clear function of the central repeat region of the malaria circumsporozoite protein has been described to date, despite its central role in the only licensed malaria vaccine. Here we use mutational analysis and single-molecule fluorescence-force spectroscopy to describe the structural properties and determine the function of this conserved region and important vaccine target.HighlightsThe CSP repeats have properties of a linear springScrambling or large truncations of the repeats leads to defects in sporozoite motilityMotility defects are attributed to abnormal formation of adhesion sites

In eukaryotes, the Origin Recognition Complex (ORC) is required for the initiation of DNA replication. The smallest subunit of ORC, Orc6, is essential for pre-replication complex (pre-RC) assembly and cell viability in yeast and for cytokinesis in metazoans. However, unlike other ORC components, the role of human Orc6 in replication remains to be resolved. Here, we identify an unexpected role for hOrc6, which is to promote S-phase progression post pre-RC assembly and DNA damage response. Orc6 localizes at the replication fork and is an accessory factor of the mismatch repair (MMR) complex. In response to oxidative damage during S-phase, often repaired by MMR, Orc6 facilitates MMR complex assembly and activity, without which the checkpoint signaling is abrogated. Mechanistically, Orc6 directly binds to MutS and enhances the chromatin-association of MutL, thus enabling efficient mismatch repair. Based on this, we conclude that hOrc6 plays a fundamental role in genome surveillance during S-phase. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC="FIGDIR/small/443400v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@bfdaa8org.highwire.dtl.DTLVardef@1ac791dorg.highwire.dtl.DTLVardef@436d1corg.highwire.dtl.DTLVardef@b07689_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIHuman Orc6 is dispensable for G1 licensing, but required for S-phase progression C_LIO_LIHuman Orc6 at the replication fork is an accessory factor for MMR complex C_LIO_LIDepletion of hOrc6 sensitizes cells to DNA damage and impairs ATR activation C_LIO_LIHuman Orc6 regulates MMR complex assembly and activity C_LI

Single-molecule fluorescence resonance energy transfer (smFRET) is a useful tool for observing the dynamics of protein-nucleic acid interactions. While the majority of smFRET measurements have used two fluorophores, multicolor smFRET measurements employing more than two fluorophores offer more information about how protein-nucleic acid complexes dynamically move, assemble, and disassemble. Multi-color smFRET experiments include three or more fluorophores and at least one donor-acceptor pair. This review highlights how multi-color smFRET is being used to probe the dynamics of three different classes of biochemical processes- protein-DNA interactions, chromatin remodeling, and protein translation.

Multi-subunit ring-ATPases carry out a myriad of biological functions, including genome packaging in viruses. Though the basic structures and functions of these motors have been well-established, the mechanisms of ATPase firing and motor coordination are poorly understood. Here, by direct counting using single-molecule fluorescence, we have determined that the active bacteriophage T4 DNA packaging motor consists of five subunits of gp17. By systematically doping motors with an ATPase-defective subunit and selecting single motors containing a precise count of active/inactive subunit(s), we found, unexpectedly, that the packaging motor can tolerate an inactive sub-unit. However, motors containing an inactive subunit(s) exhibit fewer DNA engagements, a higher failure rate in encapsidation, reduced packaging velocity, and increased pausing. These findings suggest a new packaging model in which the motor, by re-adjusting its grip on DNA, can skip an inactive subunit and resume DNA translocation, contrary to the prevailing notion of strict coordination amongst motor subunits of other packaging motors.

DNA double-strand breaks in DNA (DSBs) are common yet highly detrimental events in living organisms. To repair the damage, each cell requires a coordinated set of DNA damage response (DDR) proteins that can respond quickly, effectively, and precisely. Better understanding of these processes is therefore essential and would require an effective means of inducing targeted DSBs on demand, but previous methods are hampered by limited control over genomic location, timing, or lesion types. Tight spatiotemporal control of CRISPR-Cas9 activity has potential to overcome these limitations, which led to the development of two methods for rapid activation or deactivation of Cas9 using light. In this chapter, we discuss how control of Cas9 can advance DDR studies, describe protocols to control Cas9 activation and deactivation using this new technology, and finally outline three compatible readouts of DNA damage and the cellular response: DSB levels using droplet digital PCR, repair factor localization using ChIP-seq, and insertion-deletion (indel) repair outcomes using Sanger sequencing.

Mechanical deformations of DNA such as bending are ubiquitous and have been implicated in diverse cellular functions1. However, the lack of high-throughput tools to measure the mechanical properties of DNA has limited our understanding of how DNA mechanics influence chromatin transactions across the genome. Here we develop 'loop-seq'-a high-throughput assay to measure the propensity for DNA looping-and determine the intrinsic cyclizabilities of 270,806 50-base-pair DNA fragments that span Saccharomyces cerevisiae chromosome V, other genomic regions, and random sequences. We found sequence-encoded regions of unusually low bendability within nucleosome-depleted regions upstream of transcription start sites (TSSs). Low bendability of linker DNA inhibits nucleosome sliding into the linker by the chromatin remodeller INO80, which explains how INO80 can define nucleosome-depleted regions in the absence of other factors2. Chromosome-wide, nucleosomes were characterized by high DNA bendability near dyads and low bendability near linkers. This contrast increases for deeper gene-body nucleosomes but disappears after random substitution of synonymous codons, which suggests that the evolution of codon choice has been influenced by DNA mechanics around gene-body nucleosomes. Furthermore, we show that local DNA mechanics affect transcription through TSS-proximal nucleosomes. Overall, this genome-scale map of DNA mechanics indicates a 'mechanical code' with broad functional implications.

CRISPR-Cas9 from Streptococcus pyogenes is an RNA-guided DNA endonuclease, which has become the most popular genome editing tool. Coordinated domain motions of Cas9 prior to DNA cleavage have been extensively characterized but our understanding of Cas9 conformations postcatalysis is limited. Because Cas9 can remain stably bound to the cleaved DNA for hours, its postcatalytic conformation may influence genome editing mechanisms. Here, we use single-molecule fluorescence resonance energy transfer to characterize the HNH domain motions of Cas9 that are coupled with cleavage activity of the target strand (TS) or nontarget strand (NTS) of DNA substrate. We reveal an NTS-cleavage-competent conformation following the HNH domain conformational activation. The 3′ flap generated by NTS cleavage can be rapidly digested by a 3′ to 5′ single-stranded DNA-specific exonuclease, indicating Cas9 exposes the 3′ flap for potential interaction with the DNA repair machinery. We find evidence that the HNH domain is highly flexible post-TS cleavage, explaining a recent observation that the HNH domain was not visible in a postcatalytic cryo-EM structure. Our results illuminate previously unappreciated regulatory roles of DNA cleavage activity on Cas9’s conformation and suggest possible biotechnological applications.

The RNA-binding protein Fused in sarcoma (FUS) can form pathogenic inclusions in neurodegenerative diseases like amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD). Over 70 mutations in Fus are linked to ALS/FTLD. In patients, all Fus mutations are heterozygous, indicating that the mutant drives disease progression despite the presence of wild-type FUS. Here, we demonstrate that ALS/FTLD-linked FUS mutations in glycine (G) strikingly drive formation of droplets that do not readily interact with wild-type FUS whereas arginine (R) mutants form mixed condensates with wild-type FUS. Remarkably, interactions between wild-type and G mutants are disfavored at the earliest stages of FUS nucleation. In contrast, R mutants physically interact with the wild-type FUS such that wild-type FUS recovers the mutant defects by reducing droplet size and increasing dynamic interactions with RNA. This result suggests disparate molecular mechanisms underlying ALS/FTLD pathogenesis and differing recovery potential depending on the type of mutation.

Almost all nucleoprotein interactions and DNA manipulation events involve mechanical deformations of DNA. Extraordinary progresses in single-molecule, structural, and computational methods have characterized the average mechanical properties of DNA, such as bendability and torsional rigidity, in high resolution. Further, the advent of sequencing technology has permitted measuring, in high-throughput, how such mechanical properties vary with sequence and epigenetic modifications along genomes. We review these recent technological advancements, and discuss how they have contributed to the emerging idea that variations in the mechanical properties of DNA play a fundamental role in regulating, genome-wide, diverse processes involved in chromatin organization.

Soft 3D-fibrin-gel selected tumor repopulating cells (TRCs) from the B16F1 melanoma cell line exhibit extraordinary self-renewal and tumor-regeneration capabilities. However, their biomarkers and gene regulatory features remain largely unknown. Here, we utilized the next-generation sequencing-based RNA sequencing (RNA-seq) technique to discover novel biomarkers and active gene regulatory features of TRCs. Systems biology analysis of RNA-seq data identified differentially expressed gene clusters, including the cell adhesion cluster, which subsequently identified highly specific and novel biomarkers, such as Col2a1, Ncam1, F11r, and Negr1. We validated the expression of these genes by real-time qPCR. The expression level of Col2a1 was found to be relatively low in TRCs but twenty-fold higher compared to the parental control cell line, thus making the biomarker very specific for TRCs. We validated the COL2A1 protein by immunofluorescence microscopy, showing a higher expression of COL2A1 in TRCs compared to parental control cells. KEGG pathway analysis showed the JAK/STAT, hypoxia, and Akt signaling pathways to be active in TRCs. Besides, the aerobic glycolysis pathway was found to be very active, indicating a typical Warburg Effect on highly tumorigenic cells. Together, our study revealed highly specific biomarkers and active cell signaling pathways of melanoma TRCs that can potentially target and neutralize TRCs.

Base-pairing interactions mediate many intermolecular target recognition events. Even a single base-pair mismatch can cause a substantial difference in activity but how such changes influence the target search kinetics in vivo is unknown. Here, we use high-throughput sequencing and quantitative super-resolution imaging to probe the mutants of bacterial small RNA, SgrS, and their regulation of ptsG mRNA target. Mutations that disrupt binding of a chaperone protein, Hfq, and are distal to the mRNA annealing region still decrease the rate of target association, kon, and increase the dissociation rate, koff, showing that Hfq directly facilitates sRNA–mRNA annealing in vivo. Single base-pair mismatches in the annealing region reduce kon by 24–31% and increase koff by 14–25%, extending the time it takes to find and destroy the target by about a third. The effects of disrupting contiguous base-pairing are much more modest than that expected from thermodynamics, suggesting that Hfq buffers base-pair disruptions., Bacterial small RNA SgrS binds and regulates its primary target, ptsG mRNA. Here the authors employ Sort-Seq and super resolution imaging to investigate in vivo target recognition and rejection kinetics of SgrS.

Cas9 in complex with a programmable guide RNA targets specific double-stranded DNA for cleavage. By harnessing Cas9 as a programmable loader of superhelicase to genomic DNA, here we report a physiological-temperature DNA FISH method termed Genome Oligopaint via Local Denaturation Fluorescence in Situ Hybridization (GOLD FISH). Instead of global denaturation as in conventional DNA FISH, loading a superhelicase at a Cas9-generated nick allows for local DNA denaturation, reducing non-specific binding of probes and avoiding harsh treatments such as heat denaturation. GOLD FISH relies on Cas9 cleaving target DNA sequences and avoids the high nuclear background associated with other genome labeling methods that rely on Cas9 binding. The excellent signal brightness and specificity enable us to image non-repetitive genomic DNA loci and analyze the conformational differences between active and inactive X chromosomes. Finally, GOLD FISH could be used for rapid identification of HER2 gene amplification in a patient tissue.

Sequence features have long been known to influence the local mechanical properties and shapes of DNA. However, a mechanical code (i.e. a comprehensive mapping between DNA sequence and mechanical properties), if it exists, has been difficult to experimentally determine because direct means of measuring the mechanical properties of DNA are typically limited in throughput. Here we use Loop-seq – a recently developed technique to measure the intrinsic cyclizabilities (a proxy for bendability) of DNA fragments in genomic-scale throughput – to characterize the mechanical code. We tabulate how DNA sequence features (distribution patterns of all possible dinucleotides and dinucleotide pairs) influence intrinsic cyclizability, and build a linear model to predict intrinsic cyclizability from sequence. Using our model, we predict that DNA mechanical landscape shapes nucleosome organization around the promoters of various organisms and at the binding site of the transcription factor CTCF, and that hyperperiodic DNA inC. elegansleads to globally curved DNA segments. By performing loop-seq on random libraries in the presence or absence of CpG methylation, we show that CpG methylation leads to global stiffening of DNA in a wide sequence context, and predict based on our model that CpG methylation widely changes the mechanical landscape around mouse promoters. It suggests how epigenetic modifications of DNA might alter gene expression and mediate cellular adaptation by affecting critical processes around promoters that require mechanical deformations of DNA, such as nucleosome organization and transcription initiation. Finally, we show that the genetic code and the mechanical code are linked: sequence-dependent mechanical properties of coding DNA constrains the amino acid sequence despite the degeneracy in the genetic code. Our measurements explain why the pattern of nucleosome organization along genes influences the distribution of amino acids in the translated polypeptide.

The Hippo pathway plays an important role in developmental biology, mediating organ size by controlling cell proliferation through the activity of a core kinase cassette. Multiple upstream events activate the pathway, but how each controls this core kinase cassette is not fully understood. Activation of the core kinase cassette begins with phosphorylation of the kinase MST1/2 (also known as STK3/4). Here, using a combination of in vitro biochemistry and cell-based assays, including chemically induced dimerization and single-molecule pulldown, we revealed that increasing the proximity of adjacent kinase domains, rather than formation of a specific protein assembly, is sufficient to trigger autophosphorylation. We validate this mechanism in cells and demonstrate that multiple events associated with the active pathway, including SARAH domain–mediated homodimerization, membrane recruitment, and complex formation with the effector protein SAV1, each increase the kinase domain proximity and autophosphorylation of MST2. Together, our results reveal that multiple and distinct upstream signals each utilize the same common molecular mechanism to stimulate MST2 autophosphorylation. This mechanism is likely conserved among MST2 homologs. Our work also highlights potential differences in Hippo signal propagation between each activating event owing to differences in the dynamics and regulation of each protein ensemble that triggers MST2 autophosphorylation and possible redundancy in activation.

Mechanical deformations of DNA such as bending are ubiquitous and implicated in diverse cellular functions1. However, the lack of high-throughput tools to directly measure the mechanical properties of DNA limits our understanding of whether and how DNA sequences modulate DNA mechanics and associated chromatin transactions genome-wide. We developed an assay called loop-seq to measure the intrinsic cyclizability of DNA – a proxy for DNA bendability – in high throughput. We measured the intrinsic cyclizabilities of 270,806 50 bp DNA fragments that span the entire length of S. cerevisiae chromosome V and other genomic regions, and also include random sequences. We discovered sequence-encoded regions of unusually low bendability upstream of Transcription Start Sites (TSSs). These regions disfavor the sharp DNA bending required for nucleosome formation and are co-centric with known Nucleosome Depleted Regions (NDRs). We show biochemically that low bendability of linker DNA located about 40 bp away from a nucleosome edge inhibits nucleosome sliding into the linker by the chromatin remodeler INO80. The observation explains how INO80 can create promoter-proximal nucleosomal arrays in the absence of any other factors2 by reading the DNA mechanical landscape. We show that chromosome wide, nucleosomes are characterized by high DNA bendability near dyads and low bendability near the linkers. This contrast increases for nucleosomes deeper into gene bodies, suggesting that DNA mechanics plays a previously unappreciated role in organizing nucleosomes far from the TSS, where nucleosome remodelers predominate. Importantly, random substitution of synonymous codons does not preserve this contrast, suggesting that the evolution of codon choice has been impacted by selective pressure to preserve sequence-encoded mechanical modulations along genes. We also provide evidence that transcription through the TSS-proximal nucleosomes is impacted by local DNA mechanics. Overall, this first genome-scale map of DNA mechanics hints at a ‘mechanical code’ with broad functional implications.

Base-pairing interactions mediate intermolecular target recognition in many biological systems and applications, including DNA repair, CRISPR, microRNA, small RNA (sRNA) and antisense oligo therapies. Even a single base-pair mismatch can cause a substantial difference in biological activity but presently we do not yet know how the target search kineticsin vivoare influenced by single nucleotide level changes. Here, we used high-throughput sequencing to identify functionally relevant single point mutants of the bacterial sRNA, SgrS, and quantitative super-resolution microscopy to probe the mutational impact on the regulation of its primary target,ptsGmRNA. Our super-resolution imaging and analysis platform allowed us to further dissect mutational effects on SgrS lifetimes, and even subtle changes in thein vivorates of target association,kon, and dissociation,koff. Mutations that disrupt binding of a chaperone protein, Hfq, and are distal to the mRNA annealing region still decreasedkonand increasedkoff, providing anin vivodemonstration that Hfq directly facilitates sRNA-mRNA annealing. Single base-pair mismatches in the annealing region reducedkonby 24-31% and increasedkoffby 14-25%, extending the time it takes to find and destroy the target mRNA by about a third, depending on whether an AU or GC base-pair is disrupted. The effects of disrupting contiguous base-pairing are much more modest than that expected from thermodynamics, suggesting that Hfq also buffers base-pair disruptions.

While most SAM riboswitches strongly discriminate between SAM and SAH, the SAM/SAH riboswitch responds to both ligands with similar apparent affinities. We have determined crystal structures of the SAM/SAH riboswitch bound to SAH, SAM and other variant ligands at high resolution. The riboswitch forms an H-type pseudoknot structure with coaxial alignment of the stem–loop helix (P1) and the pseudoknot helix (PK). An additional three base pairs form at the non-open end of P1, and the ligand is bound at the interface between the P1 extension and the PK helix. The adenine nucleobase is stacked into the helix and forms a trans Hoogsteen–Watson–Crick base pair with a uridine, thus becoming an integral part of the helical structure. The majority of the specific interactions are formed with the adenosine. The methionine or homocysteine chain lies in the groove making a single hydrogen bond, and there is no discrimination between the sulfonium of SAM or the thioether of SAH. Single-molecule FRET analysis reveals that the riboswitch exists in two distinct conformations, and that addition of SAM or SAH shifts the population into a stable state that likely corresponds to the form observed in the crystal. A model for translational regulation is presented whereby in the absence of ligand the riboswitch is largely unfolded, lacking the PK helix so that translation can be initiated at the ribosome binding site. But the presence of ligand stabilizes the folded conformation that includes the PK helix, so occluding the ribosome binding site and thus preventing the initiation of translation.

G-quadruplex (G4) DNA structures can form physical barriers within the genome that must be unwound to ensure cellular genomic integrity. Here, we report unanticipated roles for the Escherichia coli Rep helicase and RecA recombinase in tolerating toxicity induced by G4-stabilizing ligands in vivo. We demonstrate that Rep and Rep-X (an enhanced version of Rep) display G4 unwinding activities in vitro that are significantly higher than the closely related UvrD helicase. G4 unwinding mediated by Rep involves repetitive cycles of G4 unfolding and refolding fueled by ATP hydrolysis. Rep-X and Rep also dislodge G4-stabilizing ligands, in agreement with our in vivo G4-ligand sensitivity result. We further demonstrate that RecA filaments disrupt G4 structures and remove G4 ligands in vitro, consistent with its role in countering cellular toxicity of G4-stabilizing ligands. Together, our study reveals novel genome caretaking functions for Rep and RecA in resolving deleterious G4 structures.

Small ribozymes such as Oryza sativa twister spontaneously cleave their own RNA when the ribozyme folds into its active conformation. The coupling between twister folding and self-cleavage has been difficult to study, however, because the active ribozyme rapidly converts to product. Here, we describe the synthesis of a photocaged nucleotide that releases guanosine within microseconds upon photosolvolysis with blue light. Application of this tool to O. sativa twister achieved the spatial (75 µm) and temporal (≤30 ms) control required to resolve folding and self-cleavage events when combined with single-molecule fluorescence detection of the ribozyme folding pathway. Real-time observation of single ribozymes after photo-deprotection showed that the precleaved folded state is unstable and quickly unfolds if the RNA does not react. Kinetic analysis showed that Mg(2+) and Mn(2+) ions increase ribozyme efficiency by making transitions to the high energy active conformation more probable, rather than by stabilizing the folded ground state or the cleaved product. This tool for light-controlled single RNA folding should offer precise and rapid control of other nucleic acid systems.

In this study, we investigated the biophysical interaction between cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and CD80. CTLA-4 is a key molecule in immunosuppression, and CD80 is a costimulatory receptor promoting T cell activation. We observed that after cell-cell contact was established between breast cancer cells and antigen presenting cells (APCs), CTLA-4 expressed on the breast cancer cells bind to CD80 expressed on the APCs, and underwent trans-endocytosis to deplete CD80. Force measurement and live cell imaging revealed that upon binding to CD80, forces generated by breast cancer cells and transmitted via CTLA-4 were sufficiently strong to displace CD80 from the surface of APCs to be internalized by breast cancer cells. We further demonstrated that because of the force-dependent trans-endocytosis of CD80, the capacity of APCs to activate T cells was significantly attenuated. Furthermore, inhibiting force generation in cancer cells would increase the T cell activating capacity of APCs. Our results provide a possible mechanism behind the immunosuppression commonly seen in breast cancer patients, and may lead to a new strategy to restore anti-tumor immunity by inhibiting pathways of force-generation.

Summary The formation of silenced and condensed heterochromatin foci involves enrichment of heterochromatin protein 1 (HP1). HP1 can bridge chromatin segments and form liquid droplets, but the biophysical principles underlying heterochromatin compartmentalization in the cell nucleus are elusive. Here, we assess mechanistically relevant features of pericentric heterochromatin compaction in mouse fibroblasts. We find that (1) HP1 has only a weak capacity to form liquid droplets in living cells; (2) the size, global accessibility, and compaction of heterochromatin foci are independent of HP1; (3) heterochromatin foci lack a separated liquid HP1 pool; and (4) heterochromatin compaction can toggle between two “digital” states depending on the presence of a strong transcriptional activator. These findings indicate that heterochromatin foci resemble collapsed polymer globules that are percolated with the same nucleoplasmic liquid as the surrounding euchromatin, which has implications for our understanding of chromatin compartmentalization and its functional consequences., Graphical Abstract, Highlights • HP1 has only a weak capacity to form droplets in living cells • Size, accessibility, and compaction of heterochromatin foci are independent of HP1 • Heterochromatin compaction is “digital” and can toggle between two distinct states • Methodological framework to assess hallmarks of phase separation in living cells, Mouse cells package heterochromatin into compact foci. Erdel et al. show that these foci lack hallmarks of liquid droplets and rather resemble collapsed polymer globules. Their size, accessibility, and compaction are independent of HP1. They can adopt two distinct folding states that possibly represent the fundamental modes of chromatin compaction.

Mechanical deformations of DNA such as bending are ubiquitous and have been implicated in diverse cellular functions

Modern genomics experiments measure functional behaviors for many thousands of DNA sequences. We suggest that, especially when these sequences are chosen at random, it is natural to compute correlation functions between sequences and measured behaviors. In simple models for the dependence of DNA flexibility on sequence, for example, correlation functions can be interpreted directly as interaction parameters. Analysis of recent experiments shows that this is surprisingly effective, leading directly to extraction of distinct features for DNA flexibility and predictions that are as accurate as more complex models. This approach follows the conventional use of correlation functions in statistical physics and connects the search for relevant DNA sequence features to the search for relevant stimulus features in the analysis of sensory neurons.

The formation of pathogenic inclusions of RNA-binding proteins in neurons is a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD). One prominent protein in these inclusions is Fused in sarcoma (FUS), and over 70 mutations in Fus are linked to ALS/FTLD. In patients, all Fus mutations are heterozygous, indicating that the mutant drives pathology despite the presence of wild-type FUS. Here, we demonstrate that ALS FUS mutations in glycine (G) strikingly drive formation of droplets that are immiscible with wild-type FUS whereas arginine (R) mutants form miscible condensates with wild-type FUS. Remarkably, immiscibility between wild-type and G mutants begins at the earliest stages of FUS nucleation. In contrast to G mutants, the miscible R mutants physically interact with the wild-type FUS such that wild-type FUS rescues the mutant defects by reducing droplet size and recovering dynamic interactions with RNA. This result suggests disparate molecular mechanisms underlying pathogenesis and differing rescue potential depending on the type of mutation.