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Abstract The outcome of CRISPR-Cas-mediated genome modifications is dependent on DNA double-strand break (DSB) processing and repair pathway choice. Homology-directed repair (HDR) of protein-blocked DSBs requires DNA end resection that is initiated by the endonuclease activity of the MRE11 complex. Using reconstituted reactions, we show that Cas9 breaks are unexpectedly not directly resectable by the MRE11 complex. In contrast, breaks catalyzed by Cas12a are readily processed. Cas9, unlike Cas12a, bridges the broken ends, preventing DSB detection and processing by MRE11. We demonstrate that Cas9 must be dislocated after DNA cleavage to allow DNA end resection and repair. Using single molecule and bulk biochemical assays, we next find that the HLTF translocase directly removes Cas9 from broken ends, which allows DSB processing by DNA end resection or non-homologous end-joining machineries. Mechanistically, the activity of HLTF requires its HIRAN domain and the release of the 3′-end generated by the cleavage of the non-target DNA strand by the Cas9 RuvC domain. Consequently, HLTF removes the H840A but not the D10A Cas9 nickase. The removal of Cas9 H840A by HLTF explains the different cellular impact of the two Cas9 nickase variants in human cells, with potential implications for gene editing.

Summary: A DNA double-strand break (DSB) jeopardizes genome integrity and endangers cell viability. Actively transcribed genes are particularly detrimental if broken and need to be repressed. However, it remains elusive how fast the repression is initiated and how far it influences the neighboring genes on the chromosome. We adopt a recently developed, very fast CRISPR to generate a DSB at a specific genomic locus with precise timing, visualize transcription in live cells, and measure the RNA polymerase II (RNAPII) occupancy near the broken site. We observe that a single DSB represses the transcription of the damaged gene in minutes, which coincides with the recruitment of a damage repair protein. Transcription repression propagates bi-directionally along the chromosome from the DSB for hundreds of kilobases, and proteasome is evoked to remove RNAPII in this process. Our method builds a foundation to measure the rapid kinetic events around a single DSB and elucidate the molecular mechanism.

The organization of nucleosomes into chromatin and their accessibility are shaped by local DNA mechanics. Conversely, nucleosome positions shape genetic variations, which may originate from mismatches during replication and chemical modification of DNA. To investigate how DNA mismatches affect the mechanical stability and the exposure of nucleosomal DNA, we used an optical trap combined with single-molecule FRET and a single-molecule FRET cyclization assay. We found that a single base-pair C-C mismatch enhances DNA bendability and nucleosome mechanical stability for the 601-nucleosome positioning sequence. An increase in force required for DNA unwrapping from the histone core is observed for single base-pair C-C mismatches placed at three tested positions: at the inner turn, at the outer turn, or at the junction of the inner and outer turn of the nucleosome. The results support a model where nucleosomal DNA accessibility is reduced by mismatches, potentially explaining the preferred accumulation of single-nucleotide substitutions in the nucleosome core and serving as the source of genetic variation during evolution and cancer progression. Mechanical stability of an intact nucleosome, that is mismatch-free, is also dependent on the species as we find that yeast nucleosomes are mechanically less stable and more symmetrical in the outer turn unwrapping compared to Xenopus nucleosomes.

Abstract The Mre11-Rad50-Nbs1 (MRN) complex recognizes and processes DNA double-strand breaks for homologous recombination by performing short-range removal of 5ʹ strands. Endonucleolytic processing by MRN requires a stably bound protein at the break site—a role we postulate is played by DNA-dependent protein kinase (DNA-PK) in mammals. Here we interrogate sites of MRN-dependent processing by identifying sites of CtIP association and by sequencing DNA-PK-bound DNA fragments that are products of MRN cleavage. These intermediates are generated most efficiently when DNA-PK is catalytically blocked, yielding products within 200 bp of the break site, whereas DNA-PK products in the absence of kinase inhibition show greater dispersal. Use of light-activated Cas9 to induce breaks facilitates temporal resolution of DNA-PK and Mre11 binding, showing that both complexes bind to DNA ends before release of DNA-PK-bound products. These results support a sequential model of double-strand break repair involving collaborative interactions between homologous and non-homologous repair complexes.

Eukaryotic gene expression is linked to chromatin structure and nucleosome positioning by ATP-dependent chromatin remodelers that establish and maintain nucleosome-depleted regions (NDRs) near transcription start sites. Conserved yeast RSC and ISW2 remodelers exert antagonistic effects on nucleosomes flanking NDRs, but the temporal dynamics of remodeler search, engagement, and directional nucleosome mobilization for promoter accessibility are unknown. Using optical tweezers and two-color single-particle imaging, we investigated the Brownian diffusion of RSC and ISW2 on free DNA and sparse nucleosome arrays. RSC and ISW2 rapidly scan DNA by one-dimensional hopping and sliding, respectively, with dynamic collisions between remodelers followed by recoil or apparent co-diffusion. Static nucleosomes block remodeler diffusion resulting in remodeler recoil or sequestration. Remarkably, both RSC and ISW2 use ATP hydrolysis to translocate mono-nucleosomes processively at ~30 bp/s on extended linear DNA under tension. Processivity and opposing push–pull directionalities of nucleosome translocation shown by RSC and ISW2 shape the distinctive landscape of promoter chromatin.

Abstract Compensatory endocytosis keeps the membrane surface area of secretory cells constant following exocytosis. At chemical synapses, clathrin-independent ultrafast endocytosis maintains such homeostasis. This endocytic pathway is temporally and spatially coupled to exocytosis; it initiates within 50 ms at the region immediately next to the active zone where vesicles fuse. However, the coupling mechanism is unknown. Here, we demonstrate that filamentous actin is organized as a ring, surrounding the active zone at mouse hippocampal synapses. Assuming the membrane area conservation is due to this actin ring, our theoretical model suggests that flattening of fused vesicles exerts lateral compression in the plasma membrane, resulting in rapid formation of endocytic pits at the border between the active zone and the surrounding actin-enriched region. Consistent with model predictions, our data show that ultrafast endocytosis requires sufficient compression by exocytosis of multiple vesicles and does not initiate when actin organization is disrupted, either pharmacologically or by ablation of the actin-binding protein Epsin1. Our work suggests that membrane mechanics underlie the rapid coupling of exocytosis to endocytosis at synapses.

In this issue of Cell Reports Methods, Molina and colleagues use in vitro single-molecule DNA flow-stretching to demonstrate the severe effects of appending a short lysine-cysteine-lysine (KCK) tag on the Bacillus subtilis ParB protein. This assay could be further utilized to evaluate the impact of other tags on DNA-binding proteins.

Visualisation of point mutations in situ is informative for studying genetic diseases. Here the authors report single guide genome oligopaint via local denaturation fluorescence in situ hybridisation, sgGOLDFISH, a direct hybridisation genome imaging method with single-nucleotide sensitivity.

Integrins come in subtypes that bind distinct ligands. This work reveals and quantifies subtype-specific mechanical force transmission and assembly of cytoskeleton architectures.

PCR is an essential method for the amplification and manipulation of nucleic acids, but the requirement for a thermocycler limits access. Here, authors engineer a helicase to replace the heating step of PCR with enzymatic unwinding, allowing the isothermal amplification of fragments up to 6 kb.

The single stranded DNA binding protein RPA coordinates DNA metabolism using multiple protein and DNA interaction domains. Here, the authors show that the chaperone-like protein Rtt105 staples RPA domains to prevent untimely protein interactions.

Signaling via the mTOR complex 1 (mTORC1) maintains cellular and organismal homeostasis. Here the authors show that the AAA + ATPase Thorase binds mTOR to promote disassembly and inactivation of mTORC1 to fine tune TOR signaling according to amino acid availability.

A new method, SENTINEL, improves the specificity of nucleic acid target sequence identification via a combination of isothermal amplification and sequence-specific DNA endonucleases. The method can discriminate single base-pair differences and takes less than 1 h to complete.

In viruses, multi-subunit ring-ATPases are involved in genome packaging. Here, using single-molecule techniques, the authors determine that the active bacteriophage T4 DNA packaging motor is a pentamer and show that the motor can tolerate inactive subunits, suggesting that strict coordination between the subunits is not crucial.

Pleckstrin homology (PH) domains are presumed to bind phosphoinositides (PIPs) but only few specific interactions are known. Using TIRF microscopy based assays, functional studies and an experimentally validated prediction algorithm, the authors show that specific PIP binding is widespread among human PH domains.

One-dimensional (1D) target search is a well-characterized phenomenon for many DNA-binding proteins but is poorly understood for chromatin remodelers. Herein, we characterize the 1D scanning properties of SWR1, a conserved yeast chromatin remodeler that performs histone exchange on +1 nucleosomes adjacent to a nucleosome-depleted region (NDR) at gene promoters. We demonstrate that SWR1 has a kinetic binding preference for DNA of NDR length as opposed to gene-body linker length DNA. Using single and dual color single-particle tracking on DNA stretched with optical tweezers, we directly observe SWR1 diffusion on DNA. We found that various factors impact SWR1 scanning, including ATP which promotes diffusion through nucleotide binding rather than ATP hydrolysis. A DNA-binding subunit, Swc2, plays an important role in the overall diffusive behavior of the complex, as the subunit in isolation retains similar, although faster, scanning properties as the whole remodeler. ATP-bound SWR1 slides until it encounters a protein roadblock, of which we tested dCas9 and nucleosomes. The median diffusion coefficient, 0.024 μm2/s, in the regime of helical sliding, would mediate rapid encounter of NDR-flanking nucleosomes at length scales found in cellular chromatin.

The functional relationship between subchondral bone and articular cartilage is unclear. Here, the authors show that transforming growth factor-beta propagates the mechanical impact of subchondral bone on articular cartilage through αV integrin–talin mechanical transduction system in chondrocytes.

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.

Many RNAs become functional before their synthesis completes. Here the authors employ a single-molecule vectorial folding assay mimicking RNA transcription and show that the ZTP riboswitch is kinetically controlled and activated by slower unwinding and strategic pausing.

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).

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.

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.

Light-up aptamers are widely used for fluorescence visualization of non-coding RNA in vivo. Here the authors employ single-molecule fluorescence-force spectroscopy to characterize the mechanical responses of the G-Quadruplex based light-up aptamers Spinach2, iMangoIII and MangoIV, which is of interest for the development of improved fluorogenic modules for imaging applications.

On arrested neutrophils a focal adhesive cluster of ~200 high affinity (HA) β2-integrin bonds under tension is sufficient to trigger Ca2+ flux that signals an increase in activation in direct proportion to increments in shear stress. We reasoned that a threshold tension acting on individual β2-integrin bonds provides a mechanical means of transducing the magnitude of fluid drag force into signals that enhance the efficiency of neutrophil recruitment and effector function. Tension gauge tethers (TGT) are a duplex of DNA nucleotides that rupture at a precise shear force, which increases with the extent of nucleotide overlap, ranging from a tolerance of 54pN to 12pN. TGT annealed to a substrate captures neutrophils via allosteric antibodies that stabilize LFA-1 in a high- or low-affinity conformation. Neutrophils sheared on TGT substrates were recorded in real time to form HA β2-integrin bonds and flux cytosolic Ca2+, which elicited shape change and downstream production of reactive oxygen species. A threshold force of 33pN triggered consolidation of HA β2-integrin bonds and triggered membrane influx of Ca2+, whereas an optimum tension of 54pN efficiently transduced activation at a level equivalent to chemotactic stimulation on ICAM-1. We conclude that neutrophils sense the level of fluid drag transduced through individual β2-integrin bonds, providing an intrinsic means to modulate inflammatory response in the microcirculation.

Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever-increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB-Dev. This position paper describes the current ‘state of the art’ from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of ‘soft recommendations’ about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage ‘open science’ practices.

Small RNAs (sRNAs) play a crucial role in the regulation of bacterial gene expression by silencing the translation of target mRNAs. SgrS is an sRNA that relieves glucose-phosphate stress, or “sugar shock” in E. coli. The power of single cell measurements is their ability to obtain population level statistics that illustrate cell-to-cell variation. Here, we utilize single molecule super-resolution microscopy in single E. coli cells coupled with stochastic modeling to analyze glucose-phosphate stress regulation by SgrS. We present a kinetic model that captures the combined effects of transcriptional regulation, gene replication and chaperone mediated RNA silencing in the SgrS regulatory network. This more complete kinetic description, simulated stochastically, recapitulates experimentally observed cellular heterogeneity and characterizes the binding of SgrS to the chaperone protein Hfq as a slow process that not only stabilizes SgrS but also may be critical in restructuring the sRNA to facilitate association with its target ptsG mRNA.

Summary: Telomeres are maintained by telomerase or in a subset of cancer cells by a homologous recombination (HR)-based mechanism, Alternative Lengthening of Telomeres (ALT). The mechanisms regulating telomere-homeostasis in ALT cells remain unclear. We report that a replication initiator protein, Origin Recognition Complex-Associated (ORCA/LRWD1), by localizing at the ALT-telomeres, modulates HR activity. ORCA's localization to the ALT-telomeres is facilitated by its interaction to SUMOylated shelterin components. The loss of ORCA in ALT-positive cells elevates the levels of two mediators of HR, RPA and RAD51, and consistent with this, we observe increased ALT-associated promyelocytic leukemia body formation and telomere sister chromatid exchange. ORCA binds to RPA and modulates the association of RPA to telomeres. Finally, the loss of ORCA causes global chromatin decondensation, including at the telomeres. Our results demonstrate that ORCA acts as an inhibitor of HR by modulating RPA binding to ssDNA and inducing chromatin compaction.

Voltage-gated sodium channels are expressed in excitable tissues and mutations have been linked to cardiac arrhythmias and channelopathies. Here the authors show that the sodium channel α-subunits interact to form a dimer and gate as dimer and that this functional dimerisation is conserved.

Ribosomes assemble through the hierarchical addition of proteins to a ribosomal RNA scaffold. Here the authors use three-color single-molecule FRET to show how the dynamics of the rRNA dictate the order in which multiple proteins assemble on the 5′ domain of the E. coli 16S rRNA.

Transcription activator-like effectors (TALEs) bind DNA through an array of tandem 34-residue repeats. How TALE repeat domains wrap around DNA, often extending more than 1.5 helical turns, without using external energy is not well understood. Here, we examine the kinetics of DNA binding of TALE arrays with varying numbers of identical repeats. Single molecule fluorescence analysis and deterministic modeling reveal conformational heterogeneity in both the free- and DNA-bound TALE arrays. Our findings, combined with previously identified partly folded states, indicate a TALE instability that is functionally important for DNA binding. For TALEs forming less than one superhelical turn around DNA, partly folded states inhibit DNA binding. In contrast, for TALEs forming more than one turn, partly folded states facilitate DNA binding, demonstrating a mode of ‘functional instability’ that facilitates macromolecular assembly. Increasing repeat number slows down interconversion between the various DNA-free and DNA-bound states.

Most replicative helicases are hexameric, ring-shaped motor proteins that translocate on and unwind DNA. Despite extensive biochemical and structural investigations, how their translocation activity is utilized chemo-mechanically in DNA unwinding is poorly understood. We examined DNA unwinding by G40P, a DnaB-family helicase, using a single-molecule fluorescence assay with a single base pair resolution. The high-resolution assay revealed that G40P by itself is a very weak helicase that stalls at barriers as small as a single GC base pair and unwinds DNA with the step size of a single base pair. Binding of a single ATPγS could stall unwinding, demonstrating highly coordinated ATP hydrolysis between six identical subunits. We observed frequent slippage of the helicase, which is fully suppressed by the primase DnaG. We anticipate that these findings allow a better understanding on the fine balance of thermal fluctuation activation and energy derived from hydrolysis.

CRISPR-Cas9 has enabled an unprecedented ability to manipulate the genome yet it is still poorly understood how target recognition functions at a molecular level. Here the authors use single-molecule FRET to probe Cas9-target interaction and identify distinct search and proofreading states.

Theoretical studies suggest that homologous DNA duplexes can preferentially associate with one another in the absence of proteins. Here, the authors show that GC-rich DNA with methylated cytosine and AT-rich DNA duplexes associate more strongly than GC-rich duplexes regardless of the sequence homology.

Cytosine modifications are important epigenetic markers yet their physical influence on DNA is not well understood. Here, Ngo et al. show that different alterations affect DNA flexibility, suggesting a mechanism where modifications change accessibility of nucleosome bound DNA.

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.

Bacteriophage T7 gp4 serves as a model protein for replicative helicases that couples deoxythymidine triphosphate (dTTP) hydrolysis to directional movement and DNA strand separation. We employed single-molecule fluorescence resonance energy transfer methods to resolve steps during DNA unwinding by T7 helicase. We confirm that the unwinding rate of T7 helicase decreases with increasing base pair stability. For duplexes containing >35% guanine-cytosine (GC) base pairs, we observed stochastic pauses every 2–3 bp during unwinding. The dwells on each pause were distributed nonexponentially, consistent with two or three rounds of dTTP hydrolysis before each unwinding step. Moreover, we observed backward movements of the enzyme on GC-rich DNAs at low dTTP concentrations. Our data suggest a coupling ratio of 1:1 between base pairs unwound and dTTP hydrolysis, and they further support the concept that nucleic acid motors can have a hierarchy of different-sized steps or can accumulate elastic energy before transitioning to a subsequent phase.

A combination of two single-molecule techniques has revealed new tertiary interactions in the TPP riboswitch.

Heterochromatic domains are enriched with repressive histone marks, including histone H3 lysine 9 methylation, written by lysine methyltransferases (KMTs). The pre-replication complex protein, origin recognition complex-associated (ORCA/LRWD1), preferentially localizes to heterochromatic regions in post-replicated cells. Its role in heterochromatin organization remained elusive. ORCA recognizes methylated H3K9 marks and interacts with repressive KMTs, including G9a/GLP and Suv39H1 in a chromatin context-dependent manner. Single-molecule pull-down assays demonstrate that ORCA-ORC (Origin Recognition Complex) and multiple H3K9 KMTs exist in a single complex and that ORCA stabilizes H3K9 KMT complex. Cells lacking ORCA show alterations in chromatin architecture, with significantly reduced H3K9 di- and tri-methylation at specific chromatin sites. Changes in heterochromatin structure due to loss of ORCA affect replication timing, preferentially at the late-replicating regions. We demonstrate that ORCA acts as a scaffold for the establishment of H3K9 KMT complex and its association and activity at specific chromatin sites is crucial for the organization of heterochromatin structure.

It is unclear that how subcellular organelles respond to external mechanical stimuli. Here, we investigated the molecular mechanisms by which mechanical force regulates Ca2+ signaling at endoplasmic reticulum (ER) in human mesenchymal stem cells. Without extracellular Ca2+, ER Ca2+ release is the source of intracellular Ca2+ oscillations induced by laser-tweezer-traction at the plasma membrane, providing a model to study how mechanical stimuli can be transmitted deep inside the cell body. This ER Ca2+ release upon mechanical stimulation is mediated not only by the mechanical support of cytoskeleton and actomyosin contractility, but also by mechanosensitive Ca2+ permeable channels on the plasma membrane, specifically TRPM7. However, Ca2+ influx at the plasma membrane via mechanosensitive Ca2+ permeable channels is only mediated by the passive cytoskeletal structure but not active actomyosin contractility. Thus, active actomyosin contractility is essential for the response of ER to the external mechanical stimuli, distinct from the mechanical regulation at the plasma membrane.

RecA proteins form a long stable filament on a single-stranded DNA and catalyze strand exchange reaction. The stability of RecA filament changes dramatically with pH, yet its detailed mechanism is not known. Here, using a single molecule assay, we determined the binding and dissociation rates of RecA monomers at the filament ends at various pH. The pH-induced rate changes were moderate but occurred in opposite directions for binding and dissociation, resulting in a substantial increase in filament stability in lower pH. The highly charged residues in C-terminal domain do not contribute to the pH dependent stability. The stability enhancement of RecA filament in low pH may help the cell to cope with acidic stress by fine-tuning of the binding and dissociation rates without losing the highly dynamic nature of the filament required for strand exchange.

Pif1 family helicases are conserved from bacteria to humans. Here, we report a novel DNA patrolling activity which may underlie Pif1’s diverse functions: a Pif1 monomer preferentially anchors itself to a 3′-tailed DNA junction and periodically reel in the 3′ tail with a step size of one nucleotide, extruding a loop. This periodic patrolling activity is used to unfold an intramolecular G-quadruplex (G4) structure on every encounter, and is sufficient to unwind RNA-DNA heteroduplex but not duplex DNA. Instead of leaving after G4 unwinding, allowing it to refold, or going beyond to unwind duplex DNA, Pif1 repeatedly unwinds G4 DNA, keeping it unfolded. Pif1-induced unfolding of G4 occurs in three discrete steps, one strand at a time, and is powerful enough to overcome G4-stabilizing drugs. The periodic patrolling activity may keep Pif1 at its site of in vivo action in displacing telomerase, resolving R-loops, and keeping G4 unfolded during replication, recombination and repair.

Progress towards a complete model of the methanogenic archaeum Methanosarcina acetivorans is reported. We characterized size distribution of the cells using differential interference contrast microscopy, finding them to be ellipsoidal with mean length and width of 2.9 μm and 2.3 μm, respectively, when grown on methanol and 30% smaller when grown on acetate. We used the single molecule pull down (SiMPull) technique to measure average copy number of the Mcr complex and ribosomes. A kinetic model for the methanogenesis pathways based on biochemical studies and recent metabolic reconstructions for several related methanogens is presented. In this model, 26 reactions in the methanogenesis pathways are coupled to a cell mass production reaction that updates enzyme concentrations. RNA expression data (RNA-seq) measured for cell cultures grown on acetate and methanol is used to estimate relative protein production per mole of ATP consumed. The model captures the experimentally observed methane production rates for cells growing on methanol and is most sensitive to the number of methyl-coenzyme-M reductase (Mcr) and methyl-tetrahydromethanopterin:coenzyme-M methyltransferase (Mtr) proteins. A draft transcriptional regulation network based on known interactions is proposed which we intend to integrate with the kinetic model to allow dynamic regulation.

Abortive cycling is a universal feature of transcription initiation catalyzed by DNA-dependent RNA polymerases (RNAP). In bacteriophage T7 RNAP, mutation of proline 266 to leucine (P266L) in the C-linker region connecting the N-terminal promoter binding domain with the C-terminal catalytic domain drastically reduces short abortive products (4-7 nt) while marginally increasing long abortives (9-11 nt). Here we have investigated the transcription initiation pathway of P266L with the goal of understanding the mechanistic basis for short and long abortive synthesis. We show that the P266L mutation does not alter the affinity for the promoter, mildly affects promoter opening, and increases the +1/+2 GTP K(d) by 2-fold. However, unlike wild-type T7 RNAP that undergoes stepwise rotation of the promoter binding domain and DNA scrunching during initial transcription, the P266L mutant does not undergo coupled rotational/scrunching movements until 7 nt RNA synthesis. The lack of rotation/scrunching correlates with greater stabilities of the initiation complexes of the P266L and decreased short abortive products. The results indicate that the increased flexibility in the C-linker due to P266L mutation enables T7 RNAP to absorb the stress from the growing RNA:DNA hybrid thereby decreasing short abortive products. Increased C-linker flexibility, however, has an adverse effect of delaying the transition into elongation by 1-2 nt, which gives rise to long abortive products. However, a mutation in the upstream promoter region greatly decreases long abortive products in P266L reactions, rendering the combination of P266L and A-15C promoter a desirable pair for efficient in vitro transcription for RNA production. We conclude that the conformational rigidity in the C-linker region conferred by the proline at position 266 is responsible for the undesirable short abortive products, but the rigidity is critical for efficient promoter clearance and transition into elongation.

The telomere-ending binding protein complex CST (Cdc13-Stn1-Ten1) mediates critical functions in both telomere protection and replication. We devised a co-expression and affinity purification strategy for isolating large quantities of the complete Candida glabrata CST complex. The complex was found to exhibit a 2∶4∶2 or 2∶6∶2 stoichiometry as judged by the ratio of the subunits and the native size of the complex. Stn1, but not Ten1 alone, can directly and stably interact with Cdc13. In gel mobility shift assays, both Cdc13 and CST manifested high-affinity and sequence-specific binding to the cognate telomeric repeats. Single molecule FRET-based analysis indicates that Cdc13 and CST can bind and unfold higher order G-tail structures. The protein and the complex can also interact with non-telomeric DNA in the absence of high-affinity target sites. Comparison of the DNA-protein complexes formed by Cdc13 and CST suggests that the latter can occupy a longer DNA target site and that Stn1 and Ten1 may contact DNA directly in the full CST-DNA assembly. Both Stn1 and Ten1 can be cross-linked to photo-reactive telomeric DNA. Mutating residues on the putative DNA-binding surface of Candida albicans Stn1 OB fold domain caused a reduction in its crosslinking efficiency in vitro and engendered long and heterogeneous telomeres in vivo, indicating that the DNA-binding activity of Stn1 is required for telomere protection. Our data provide insights on the assembly and mechanisms of CST, and our robust reconstitution system will facilitate future biochemical analysis of this important complex.

Integrins, a class of membrane proteins involved in cell adhesion, participate in the cell’s sensing of the mechanical environments. We previously showed that, for the initial cell adhesion to occur, single integrins need to experience a threshold force of 40 pico-Newton (pN) through their bond with surface-bound ligands. This force requirement was determined using a series of double-stranded DNA tethers called tension gauge tethers (TGTs), each with a different rupture force, linked to the ligand. Here, we performed cell-adhesion experiments using surfaces coated with two different TGTs, one of a strong rupture force (around 54 pN) and the other of a weak rupture force (around 12 pN). When presented with one type of TGT only, cells adhered to the strong TGT-coated surface but not to the weak TGT-coated surface. However, when presented with both, the presence of the strong TGTs transforms the way cells respond to the weak TGTs such that cells treat both TGTs the same, as if the weak TGTs were strong. Furthermore, a subpopulation of cells can adhere to and spread on a surface displaying just a few molecules of the strong TGTs per cell if, and only if, they are presented along with many weak TGTs. This ultrasensitivity to just a few tethers that can withstand strong forces raises a question of how the cells can achieve such remarkable sensitivity to their mechanical environment without amplifying noise.

During homologous recombination, RecA forms a helical filament on a single stranded (ss) DNA that searches for a homologous double stranded (ds) DNA and catalyzes the exchange of complementary base pairs to form a new heteroduplex. Using single molecule fluorescence imaging tools with high spatiotemporal resolution we characterized the encounter complex between the RecA filament and dsDNA. We present evidence in support of the ‘sliding model’ wherein a RecA filament diffuses along a dsDNA track. We further show that homology can be detected during sliding. Sliding occurs with a diffusion coefficient of approximately 8000 bp2/s allowing the filament to sample several hundred base pairs before dissociation. Modeling suggests that sliding can accelerate homology search by as much as 200 fold. Homology recognition can occur for as few as 6 nt of complementary basepairs with the recognition efficiency increasing for higher complementarity. Our data represents the first example of a DNA bound multi-protein complex which can slide along another DNA to facilitate target search.

Complex viruses are assembled from simple protein subunits by sequential and irreversible assembly. During genome packaging in bacteriophages, a powerful molecular motor assembles at the special portal vertex of an empty prohead to initiate packaging. The capsid expands after about 10%-25% of the genome is packaged. When the head is full, the motor cuts the concatemeric DNA and dissociates from the head. Conformational changes, particularly in the portal, are thought to drive these sequential transitions. We found that the phage T4 packaging machine is highly promiscuous, translocating DNA into finished phage heads as well as into proheads. Optical tweezers experiments show that single motors can force exogenous DNA into phage heads at the same rate as into proheads. Single molecule fluorescence measurements demonstrate that phage heads undergo repeated initiations, packaging multiple DNA molecules into the same head. These results suggest that the phage DNA packaging machine has unusual conformational plasticity, powering DNA into an apparently passive capsid receptacle, including the highly stable virus shell, until it is full. These features probably led to the evolution of viral genomes that fit capsid volume, a strikingly common phenomenon in double-stranded DNA viruses, and will potentially allow design of a novel class of nanocapsid delivery vehicles.