Single-molecule Tethered Particle Motion Research Articles

DNA-bridging by an archaeal histone variant via a unique tetramerisation interface

In eukaryotes, histone paralogues form obligate heterodimers such as H3/H4 and H2A/H2B that assemble into octameric nucleosome particles. Archaeal histones are dimeric and assemble on DNA into ‘hypernucleosome’ particles of varying sizes with each dimer wrapping 30 bp of DNA. These are composed of canonical and variant histone paralogues, but the function of these variants is poorly understood. Here, we characterise the structure and function of the histone paralogue MJ1647 from Methanocaldococcus jannaschii that has a unique C-terminal extension enabling homotetramerisation. The 1.9 Å X-ray structure of a dimeric MJ1647 species, structural modelling of the tetramer, and site-directed mutagenesis reveal that the C-terminal tetramerization module consists of two alpha helices in a handshake arrangement. Unlike canonical histones, MJ1647 tetramers can bridge two DNA molecules in vitro. Using single-molecule tethered particle motion and DNA binding assays, we show that MJ1647 tetramers bind ~60 bp DNA and compact DNA in a highly cooperative manner. We furthermore show that MJ1647 effectively competes with the transcription machinery to block access to the promoter in vitro. To the best of our knowledge, MJ1647 is the first histone shown to have DNA bridging properties, which has important implications for genome structure and gene expression in archaea.

Microcephaly family protein MCPH1 stabilizes RAD51 filaments.

Microcephalin 1 (MCPH1) was identified from genetic mutations in patients with primary autosomal recessive microcephaly. In response to DNA double-strand breaks (DSBs), MCPH1 forms damage-induced foci and recruits BRCA2–RAD51 complex, a key component of the DSB repair machinery for homologous recombination (HR), to damage sites. Accordingly, the efficiency of HR is significantly attenuated upon depletion of MCPH1. The biochemical characteristics of MCPH1 and its functional interaction with the HR machinery had remained unclear due to lack of highly purified MCPH1 recombinant protein for functional study. Here, we established a mammalian expression system to express and purify MCPH1 protein. We show that MCPH1 is a bona fide DNA-binding protein and provide direct biochemical analysis of this MCPH family protein. Furthermore, we reveal that MCPH1 directly interacts with RAD51 at multiple contact points, providing evidence for how MCPH1 physically engages with the HR machinery. Importantly, we demonstrate that MCPH1 enhances the stability of RAD51 on single-strand DNA, a prerequisite step for RAD51-mediated recombination. Single-molecule tethered particle motion analysis showed a ∼2-fold increase in the lifetime of RAD51–ssDNA filaments in the presence of MCPH1. Thus, our study demonstrates direct crosstalk between microcephaly protein MCPH1 and the recombination component RAD51 for DSB repair.

Rad51 facilitates filament assembly of meiosis-specific Dmc1 recombinase

Dmc1 recombinases are essential to homologous recombination in meiosis. Here, we studied the kinetics of the nucleoprotein filament assembly of Saccharomyces cerevisiae Dmc1 using single-molecule tethered particle motion experiments and in vitro biochemical assay. ScDmc1 nucleoprotein filaments are less stable than the ScRad51 ones because of the kinetically much reduced nucleation step. The lower nucleation rate of ScDmc1 results from its lower single-stranded DNA (ssDNA) affinity, compared to that of ScRad51. Surprisingly, ScDmc1 nucleates mostly on the DNA structure containing the single-stranded and duplex DNA junction with the allowed extension in the 5'-to-3' polarity, while ScRad51 nucleation depends strongly on ssDNA lengths. This nucleation preference is also conserved for mammalian RAD51 and DMC1. In addition, ScDmc1 nucleation can be stimulated by short ScRad51 patches, but not by EcRecA ones. Pull-down experiments also confirm the physical interactions of ScDmc1 with ScRad51 in solution, but not with EcRecA. Our results are consistent with a model that Dmc1 nucleation can be facilitated by a structural component (such as DNA junction and protein-protein interaction) and DNA polarity. They provide direct evidence of how Rad51 is required for meiotic recombination and highlight a regulation strategy in Dmc1 nucleoprotein filament assembly.

Polyamines stimulate RecA-mediated recombination by condensing duplex DNA and stabilizing intermediates.

Polyamines are naturally occurring cationic molecules in cells. In addition to their roles in modulating gene expression and cell proliferation, they have been shown to stimulate DNA recombination. The molecular mechanism for stimulation is not clear. We utilized single-molecule tethered particle motion (TPM) experiments to investigate how polyamines stimulate RecA-mediated recombination. We showed that natural polyamines, spermine and spermidine, condense duplex DNA, but with different efficiencies. While ∼300 μM of spermine condenses 50% of duplex DNA, 2.0 mM of spermidine is required to achieve the same level of condensation. The condensation takes place in a stepwise manner, and is reversible upon removal of polyamines. We also showed that addition of polyamines stimulates the duplex capture activity of RecA filament and stabilizes the intermediates with longer dwell time. Through condensing duplex DNA and stabilizing the complex of RecA filaments and duplex DNA, polyamines stimulate the formation of functional intermediates by ∼20-fold, and promote recombination progression.

A 5'-to-3' strand exchange polarity is intrinsic to RecA nucleoprotein filaments in the absence of ATP hydrolysis.

RecA is essential to recombinational DNA repair in which RecA filaments mediate the homologous DNA pairing and strand exchange. Both RecA filament assembly and the subsequent DNA strand exchange are directional. Here, we demonstrate that the polarity of DNA strand exchange is embedded within RecA filaments even in the absence of ATP hydrolysis, at least over short DNA segments. Using single-molecule tethered particle motion, we show that successful strand exchange in the presence of ATP proceeds with a 5′-to-3′ polarity, as demonstrated previously. RecA filaments prepared with ATPγS also exhibit a 5′-to-3′ progress of strand exchange, suggesting that the polarity is not determined by RecA disassembly and/or ATP hydrolysis. RecAΔC17 mutants, lacking a C-terminal autoregulatory flap, also promote strand exchange in a 5′-to-3′ polarity in ATPγS, a polarity that is largely lost with this RecA variant when ATP is hydrolyzed. We propose that there is an inherent strand exchange polarity mediated by the structure of the RecA filament groove, associated by conformation changes propagated in a polar manner as DNA is progressively exchanged. ATP hydrolysis is coupled to polar strand exchange over longer distances, and its contribution to the polarity requires an intact RecA C-terminus.

The role of near-wall drag effects in the dynamics of tethered DNA under shear flow

We utilized single-molecule tethered particle motion (TPM) tracking, optimized for studying the behavior of short (0.922 μm) dsDNA molecules under shear flow conditions, in the proximity of a wall (surface). These experiments track the individual trajectories through a gold nanobead (40 nm in radius), attached to the loose end of the DNA molecules. Under such circumstances, local interactions with the wall become more pronounced, manifested through hydrodynamic interactions. To elucidate the mechanical mechanism that affects the statistics of the molecular trajectories of the tethered molecules, we estimate the resting diffusion coefficient of our system. Using this value and our measured data, we calculate the orthogonal distance of the extended DNA molecules from the surface. This calculation considers the hydrodynamic drag effect that emerges from the proximity of the molecule to the surface, using the Faxén correction factors. Our finding enables the construction of a scenario according to which the tension along the chain builds up with the applied shear force, driving the loose end of the DNA molecule away from the wall. With the extension from the wall, the characteristic times of the system decrease by three orders of magnitude, while the drag coefficients decay to a plateau value that indicates that the molecule still experiences hydrodynamic effects due to its proximity to the wall.

DNA with Different Local Torsional States Affects RecA-Mediated Recombination Progression.

DNA topology is thought to affect DNA enzyme activity. The helical structure of duplex DNA dictates the change of topological states during strand separation when DNA is constrained. During the repair of DNA double-stranded breaks, the RecA nucleoprotein filament invades DNA and carries out consecutive strand exchange reactions coupled with duplex DNA strand separation. It has been suggested that torsional strain could be generated and its accumulation could inhibit strand exchange. We used hairpin and nicked DNA substrates to test how torsional strain alters the RecA-mediated strand exchange efficiency. Single-molecule tethered particle motion (TPM) experiments showed that torsionally constrained hairpin DNA substrates returned nearly no successful strand exchange events catalyzed by RecA. Surprisingly, the strand exchange efficiencies increase in the presence of DNA nicks or loop disruption. The dwell time of transient RecA events in hairpin is shorter compared to those found in nicked or fork DNA substrates, which suggests a limited strand exchange progression in hairpin substrates. Our observation shows that RecA generates local torsional strain during strand exchange, and the inability to dissipate this torsional strain inhibits homologous recombination progression. DNA topological states are thus important regulation measures of DNA recombination.

Single-molecule analysis of ϕC31 integrase-mediated site-specific recombination by tethered particle motion.

Serine and tyrosine site-specific recombinases (SRs and YRs, respectively) provide templates for understanding the chemical mechanisms and conformational dynamics of strand cleavage/exchange between DNA partners. Current evidence suggests a rather intriguing mechanism for serine recombination, in which one half of the cleaved synaptic complex undergoes a 180° rotation relative to the other. The ‘small’ and ‘large’ SRs contain a compact amino-terminal catalytic domain, but differ conspicuously in their carboxyl-terminal domains. So far, only one serine recombinase has been analyzed using single substrate molecules. We now utilized single-molecule tethered particle motion (TPM) to follow step-by-step recombination catalyzed by a large SR, phage ϕC31 integrase. The integrase promotes unidirectional DNA exchange between attB and attP sites to integrate the phage genome into the host chromosome. The recombination directionality factor (RDF; ϕC31 gp3) activates the excision reaction (attL × attR). From integrase-induced changes in TPM in the presence or absence of gp3, we delineated the individual steps of recombination and their kinetic features. The gp3 protein appears to regulate recombination directionality by selectively promoting or excluding active conformations of the synapse formed by specific att site partners. Our results support a ‘gated rotation’ of the synaptic complex between DNA cleavage and joining.

Probing a Label-Free Local Bend in DNA by Single Molecule Tethered Particle Motion

Being capable of characterizing DNA local bending is essential to understand thoroughly many biological processes because they involve a local bending of the double helix axis, either intrinsic to the sequence or induced by the binding of proteins. Developing a method to measure DNA bend angles that does not perturb the conformation of the DNA itself or the DNA-protein complex is a challenging task. Here, we propose a joint theory-experiment high throughput approach to rigorously measure such bend angles using the Tethered Particle Motion (TPM) technique. By carefully modeling the TPM geometry, we propose a simple formula based on a kinked Worm-Like Chain model to extract the bend angle from TPM measurements. Using constructs made of 575 base-pair DNAs with in-phase assemblies of 1 to 7 6A-tracts, we find that the sequence CA6CGG induces a bend angle of 19 [4] {\deg}. Our method is successfully compared to more theoretically complex or experimentally invasive ones such as cyclization, NMR, FRET or AFM. We further apply our procedure to TPM measurements from the literature and demonstrate that the angles of bends induced by proteins, such as Integration Host Factor (IHF) can be reliably evaluated as well.

Probing a label-free local bend in DNA by single molecule tethered particle motion.

Being capable of characterizing DNA local bending is essential to understand thoroughly many biological processes because they involve a local bending of the double helix axis, either intrinsic to the sequence or induced by the binding of proteins. Developing a method to measure DNA bend angles that does not perturb the conformation of the DNA itself or the DNA-protein complex is a challenging task. Here, we propose a joint theory-experiment high-throughput approach to rigorously measure such bend angles using the Tethered Particle Motion (TPM) technique. By carefully modeling the TPM geometry, we propose a simple formula based on a kinked Worm-Like Chain model to extract the bend angle from TPM measurements. Using constructs made of 575 base-pair DNAs with in-phase assemblies of one to seven 6A-tracts, we find that the sequence CA6CGG induces a bend angle of 19° ± 4°. Our method is successfully compared to more theoretically complex or experimentally invasive ones such as cyclization, NMR, FRET or AFM. We further apply our procedure to TPM measurements from the literature and demonstrate that the angles of bends induced by proteins, such as Integration Host Factor (IHF) can be reliably evaluated as well.

Single molecule TPM analysis of the catalytic pentad mutants of Cre and Flp site-specific recombinases: contributions of the pentad residues to the pre-chemical steps of recombination.

Cre and Flp site-specific recombinase variants harboring point mutations at their conserved catalytic pentad positions were characterized using single molecule tethered particle motion (TPM) analysis. The findings reveal contributions of these amino acids to the pre-chemical steps of recombination. They suggest functional differences between positionally conserved residues in how they influence recombinase-target site association and formation of ‘non-productive’, ‘pre-synaptic’ and ‘synaptic’ complexes. The most striking difference between the two systems is noted for the single conserved lysine. The pentad residues in Cre enhance commitment to recombination by kinetically favoring the formation of pre-synaptic complexes. These residues in Flp serve a similar function by promoting Flp binding to target sites, reducing non-productive binding and/or enhancing the rate of assembly of synaptic complexes. Kinetic comparisons between Cre and Flp, and between their derivatives lacking the tyrosine nucleophile, are consistent with a stronger commitment to recombination in the Flp system. The effect of target site orientation (head-to-head or head-to-tail) on the TPM behavior of synapsed DNA molecules supports the selection of anti-parallel target site alignment prior to the chemical steps. The integrity of the synapse, whose establishment/stability is fostered by strand cleavage in the case of Flp but not Cre, appears to be compromised by the pentad mutations.

Histone chaperone-mediated nucleosome assembly process.

A huge amount of information is stored in genomic DNA and this stored information resides inside the nucleus with the aid of chromosomal condensation factors. It has been reported that the repeat nucleosome core particle (NCP) consists of 147-bp of DNA and two copies of H2A, H2B, H3 and H4. Regulation of chromosomal structure is important to many processes inside the cell. In vivo, a group of histone chaperones facilitate and regulate nucleosome assembly. How NCPs are constructed with the aid of histone chaperones remains unclear. In this study, the histone chaperone-mediated nucleosome assembly process was investigated using single-molecule tethered particle motion (TPM) experiments. It was found that Asf1 is able to exert more influence than Nap1 and poly glutamate acid (PGA) on the nucleosome formation process, which highlights Asf1’s specific role in tetrasome formation. Thermodynamic parameters supported a model whereby energetically favored nucleosomal complexes compete with non-nucleosomal complexes. In addition, our kinetic findings propose the model that histone chaperones mediate nucleosome assembly along a path that leads to enthalpy-favored products with free histones as reaction substrates.

Estimation of DNA Loop Interactions Supports the Loop Domain Model of Insulator Action

Long range regulation of transcription by regulatory proteins that bind to DNA elements involves DNA-looping interactions. The high specificity of these interactions is not well understood but may result from the “loop domain model”. According to this model, insulators form DNA loops and partition DNA into separate topological domains. Interactions between DNA elements within domains are enhanced while those between domains are attenuated. To quantify the specificity of DNA-loop interactions, DNA constructs were designed to form separate or overlapping loops mediated by lac and lambda repressors. DNA constructs with identical loop segments were used in both in vivo studies in E. coli and in vitro single molecule tethered particle motion (TPM) experiments. In the presence of lac and/or lambda repressors, the probabilities of loop formation ‘within’ or ‘between’ topological domains established by another loop were quantified. Fitting the probabilities to a statistical model produced an estimate of the assistance or interference that formation of one loop provided to a second loop forming within or across the boundaries of the first loop. Between domains loop interference was calculated to be 10 fold in vivo 2.5 fold in vitro. In contrast within domains, assistance was calculated to be 3 fold in vivo and 12.6 fold in vitro. Supercoiling within bacteria might be the reason for the difference between in vivo and in vitro estimates. Modelling these data shows that a combination of interference between domains and assistance within domains can generate highly specific long range interactions.

Mutations altering the interplay between GkDnaC helicase and DNA reveal an insight into helicase unwinding (LB125)

DNA helicase is a motor protein that unwinds and separates duplex DNA using energy derived from the hydrolysis of nucleoside triphosphates (NTPs). In different organisms, helicase has a number of different mechanisms for unwinding activity. The studies of the helicase activity in complex with individual and sets of accessory proteins will elucidate the complexity of the regulatory network within the whole replisome machinery. In this study, we use the replicative helicase of Geobacillus kaustophilus, a gram‐positive bacterium, as a model system (GkDnaC). Based on our previous crystal structure of the GkDnaC‐ssDNA complex, we identified several residues involved in ssDNA binding. The locations of these key residues imply that these ssDNA‐interacting sites correlate with helicase translocation. To investigate the nature of unwinding by hexameric GkDnaC helicase (superfamilies IV), we studied how the activity of GkDnaC is modulated by specific mutations and by accessory proteins. Our results imply that a partial loss in the interaction of helicase with ssDNA leads to an enhancement in helicase activity, while their ATPase activities remain unchanged. Based on kinetic evidences from single‐molecule tethered particle motion (smTPM) experiments, the unwinding rates of these GkDnaC mutants were consistently faster than that of wild‐type enzyme. In addition, we characterized the role of accessory proteins of GkDnaC helicase, showing that the interaction with accessory proteins alters both the ATPase and the unwinding velocity of the helicase.

Tethered Particle Motion Reveals that LacI·DNA Loops Coexist with a Competitor-Resistant but Apparently Unlooped Conformation

The lac repressor protein (LacI) efficiently represses transcription of the lac operon in Escherichia coli by binding to two distant operator sites on the bacterial DNA and causing the intervening DNA to form a loop. We employed single-molecule tethered particle motion to observe LacI-mediated loop formation and breakdown in DNA constructs that incorporate optimized operator binding sites and intrinsic curvature favorable to loop formation. Previous bulk competition assays indirectly measured the loop lifetimes in these optimized DNA constructs as being on the order of days; however, we measured these same lifetimes to be on the order of minutes for both looped and unlooped states. In a range of single-molecule DNA competition experiments, we found that the resistance of the LacI-DNA complex to competitive binding is a function of both the operator strength and the interoperator sequence. To explain these findings, we present what we believe to be a new kinetic model of loop formation and DNA competition. In this proposed new model, we hypothesize a new unlooped state in which the unbound DNA-binding domain of the LacI protein interacts nonspecifically with nonoperator DNA adjacent to the operator site at which the second LacI DNA-binding domain is bound.

RecBCD Fails to Bypass the 5′-To-3′ Single-Stranded DNA Gap After Translocating Along Individual Chi-Containing Duplex DNA

The E. coli RecBCD plays an important role of initiating the repair of double-stranded DNA breaks (DSB). Translocating and recognizing chi sequence (5'-GCTGGTGG-3') have been implicated with a conformational change that enables the enzyme to preserve the 3'-to-5' single-stranded DNA for RecA assembly. RecBCD is composed of three subunits, RecB (3'-to-5' helicase), RecD (5'-to-3' helicase), and RecC. Here we used a single-molecule tethered particle motion technique to directly monitor the translocation of RecBCD along chi-contained DNA molecules. Using bead-labeled enzymes, we monitored the RecBCD translocation along individual DNA by measuring the gradual decrease in the bead Brownian motion as the enzyme moves along the DNA towards the surface. DNA substrates were designed that RecBCD would encounter a single-stranded DNA gap after the recognition of the chi sequence. While translocating along chi-free DNA substrates, the time traces showed no apparent pause, which neither 3'-to-5' nor 5'-to-3' ssDNA gap influents the movement of the enzyme1. However, over 50% of RecBCD enzymes failed to pass through the 5'-to-3' ssDNA gap after translocating over chi-containing duplex DNA. Considering RecD as a major 5'-to-3' ssDNA translocase in the RecBCD complex, our observation is consistent with the model that the conformation change occurs after chi recognition and RecD is disengaged from the 5'-ssDNA.(1) Chung, C.; Li, H. W. J. Am. Chem. Soc. 2013, 135, 8920.

Pif1 Regulates Telomere Length by Removing Telomerase from Telomere Ends

The repetitive TG-rich DNA sequence at the chromosome end, telomere, protects cells from the end-replication problem and abnormal DNA degradation in eukaryotic cells. The maintenance of telomere length is responsible for aging as well as cancer cell proliferation. Saccharomyces cerevisiae telomerase (Est2) is the reverse transcriptase responsible for extending telomeres in yeast. Pif1 helicase has been implicated in regulating the telomerase activity. We used single-molecule tethered particle motion experiments to directly investigate how Pif1 helicases regulate telomerase activity. We found that Est2 telomerase stayed bound to the telomere end after extension, but Pif1 helicases remove telomerase from the telomere. In the presence of Pif1 helicases, multiple runs of the telomerase-mediated telomere lengthening were observed. This suggests a model that Pif1 helicases remove telomerase from the telomere ends, allowing the telomerase recycling. We also observed that the ssDNA gap size affects the telomerase removal efficiency by Pif1 helicases, but not for the helicase unwinding efficiency. It is likely that efficient removal of telomerase from the telomere end is favored by the presence of multiple encountering of Pif1 helicases.

Organization of DNA Partners and Strand Exchange Mechanisms during Flp Site-Specific Recombination Analyzed by Difference Topology, Single Molecule FRET and Single Molecule TPM

Flp site-specific recombination between two target sites (FRTs) harboring non-homology within the strand exchange region does not yield stable recombinant products. In negatively supercoiled plasmids containing head-to-tail sites, the reaction produces a series of knots with odd-numbered crossings. When the sites are in head-to-head orientation, the knot products contain even-numbered crossings. Both types of knots retain parental DNA configuration. By carrying out Flp recombination after first assembling the topologically well defined Tn3 resolvase synapse, it is possible to determine whether these knots arise by a processive or a dissociative mechanism. The nearly exclusive products from head-to-head and head-to-tail oriented “non-homologous” FRT partners are a 4-noded knot and a 5-noded knot, respectively. The corresponding products from a pair of native (homologous) FRT sites are a 3-noded knot and a 4-noded catenane, respectively. These results are consistent with non-homology-induced two rounds of dissociative recombination by Flp, the first to generate reciprocal recombinants containing non-complementary base pairs and the second to produce parental molecules with restored base pairing. Single molecule fluorescence resonance energy transfer (smFRET) analysis of geometrically restricted FRTs, together with single molecule tethered particle motion (smTPM) assays of unconstrained FRTs, suggests that the sites are preferentially synapsed in an anti-parallel fashion. This selectivity in synapse geometry occurs prior to the chemical steps of recombination, signifying early commitment to a productive reaction path. The cumulative topological, smFRET and smTPM results have implications for the relative orientation of DNA partners and the directionality of strand exchange during recombination mediated by tyrosine site-specific recombinases.

Using Single-Molecule Approaches To Study Archaeal DNA-Binding Protein Alba1

Thermophilic and hyperthermophilic archaea have one or more copies of the Alba gene, which encodes Alba, a dimeric, highly basic protein that binds cooperatively to DNA. However, the functions of Alba and how it interacts with DNA remain unclear. In this study, we have used single-molecule tethered particle motion (TPM) and optical tweezers (OT) experiments to study the interactions between DNA molecules and Alba1. When Alba1 binds to double-stranded DNA, the Brownian motion (BM) amplitude for DNA tethers increases continuously, suggesting that Alba1 binds cooperatively. The OT study confirmed that a 5-fold increase in the persistence length of the Alba1 nucleoprotein filament is the major factor causing the increase in the BM amplitude for DNA tethers, while the contour length remained mostly unchanged. Moreover, the rate of the increase in the BM amplitude and the BM plateau value are both DNA length-dependent, indicating that the number of Alba1 initiation binding sites increases as the DNA becomes longer. Using the incoming-strand TPM experiment to monitor the interaction between Alba1 nucleoprotein filaments, we found that significant dimer-dimer contacts between two Alba1 nucleoprotein filaments are present, and the interaction is regulated by the concentration of Alba1.

Poly(dA:dT)-Rich DNAs Are Highly Flexible in the Context of DNA Looping

Large-scale DNA deformation is ubiquitous in transcriptional regulation in prokaryotes and eukaryotes alike. Though much is known about how transcription factors and constellations of binding sites dictate where and how gene regulation will occur, less is known about the role played by the intervening DNA. In this work we explore the effect of sequence flexibility on transcription factor-mediated DNA looping, by drawing on sequences identified in nucleosome formation and ligase-mediated cyclization assays as being especially favorable for or resistant to large deformations. We examine a poly(dA:dT)-rich, nucleosome-repelling sequence that is often thought to belong to a class of highly inflexible DNAs; two strong nucleosome positioning sequences that share a set of particular sequence features common to nucleosome-preferring DNAs; and a CG-rich sequence representative of high G+C-content genomic regions that correlate with high nucleosome occupancy in vivo. To measure the flexibility of these sequences in the context of DNA looping, we combine the in vitro single-molecule tethered particle motion assay, a canonical looping protein, and a statistical mechanical model that allows us to quantitatively relate the looping probability to the looping free energy. We show that, in contrast to the case of nucleosome occupancy, G+C content does not positively correlate with looping probability, and that despite sharing sequence features that are thought to determine nucleosome affinity, the two strong nucleosome positioning sequences behave markedly dissimilarly in the context of looping. Most surprisingly, the poly(dA:dT)-rich DNA that is often characterized as highly inflexible in fact exhibits one of the highest propensities for looping that we have measured. These results argue for a need to revisit our understanding of the mechanical properties of DNA in a way that will provide a basis for understanding DNA deformation over the entire range of biologically relevant scenarios that are impacted by DNA deformability.