DNA Bending Research Articles

Dynamics of Target DNA Binding and Cleavage by Staphylococcus aureus Cas9 as Revealed by High-Speed Atomic Force Microscopy.

Programmable DNA binding and cleavage by CRISPR-Cas9 has revolutionized the life sciences. However, the off-target cleavage observed in DNA sequences with some homology to the target still represents a major limitation for a more widespread use of Cas9 in biology and medicine. For this reason, complete understanding of the dynamics of DNA binding, interrogation and cleavage by Cas9 is crucial to improve the efficiency of genome editing. Here, we use high-speed atomic force microscopy (HS-AFM) to investigate Staphylococcus aureus Cas9 (SaCas9) and its dynamics of DNA binding and cleavage. Upon binding to single-guide RNA (sgRNA), SaCas9 forms a close bilobed structure that transiently and flexibly adopts also an open configuration. The SaCas9-mediated DNA cleavage is characterized by release of cleaved DNA and immediate dissociation, confirming that SaCas9 operates as a multiple turnover endonuclease. According to present knowledge, the process of searching for target DNA is mainly governed by three-dimensional diffusion. Independent HS-AFM experiments show a potential long-range attractive interaction between SaCas9-sgRNA and its target DNA. The interaction precedes the formation of the stable ternary complex and is observed exclusively in the vicinity of the protospacer-adjacent motif (PAM), up to distances of several nanometers. The direct visualization of the process by sequential topographic images suggests that SaCas9-sgRNA binds to the target sequence first, while the following binding of the PAM is accompanied by local DNA bending and formation of the stable complex. Collectively, our HS-AFM data reveal a potential and unexpected behavior of SaCas9 during the search for DNA targets.

Fluidification of Entanglements by a DNA Bending Protein

In spite of the nanoscale and single-molecule insights into nucleoid associated proteins (NAPs), their role in modulating the mesoscale viscoelasticity of entangled DNA has been overlooked so far. By combining microrheology and molecular dynamics simulation, we find that the abundant NAP "integration host factor" (IHF) lowers the viscosity of entangled λDNA 20-fold at physiological concentrations and stoichiometries. Our results suggest that IHF may play a previously unappreciated role in resolving DNA entanglements and in turn may be acting as a "genomic fluidizer" for bacterial genomes.

DNA bending facilitates the formation of Z-DNA investigated by single-molecule FRET.

Z-DNA, a noncanonical helical structure of double-stranded DNA (dsDNA), plays essential roles in diverse biological processes such as transcription regulation. Mechanical stresses on dsDNA, such as torsion and tension, could form Z-DNA more easily. However, the effect of DNA bending, one of the most common deformations of DNA, on Z-DNA formation is utterly elusive. Herein, we show that DNA bending facilitates the formation of Z-DNA, that is, Z-DNA is formed at lower Mg2+ concentration as the bending force becomes stronger. We regulated the bending force on dsDNA via D-shaped DNA nanostructures and observed the B-Z transition by single-molecule FRET. In the presence of the bending force, the B-Z transition of methylated DNA occurred at a 28-fold lower Mg2+ condition (2.8 mM) which is in the range of physiological salt concentrations. Monte Carlo simulation suggested that the base extrusion at the B-Z junction according to the B-Z transition may stabilize the Z-DNA form via releasing the bending stress on dsDNA. Our results certainly show that DNA bending force induces the formation of Z-DNA and provide a new insight into the effect of DNA bending.

Uracil-DNA glycosylase efficiency is modulated by substrate rigidity.

DNA sequence effects have been identified for many glycosylases and alkyl-transferases that repair base mismatches, uracil bases, alkylated bases, etc. Here, we focused on understanding how DNA sequence impacts the repair of uracil, a highly mutagenic and common lesion in DNA. Uracil DNA-glycosylase (UNG) is a base excision repair enzyme that removes uracil from DNA by a base flipping mechanism. UNG excision efficiency depends on DNA sequence. We show that UNG efficiency is dictated by the intrinsic local deformability of the substrate sequence around the uracil. UNG specificity constants (kcat/KM) and DNA flexibilities were measured for an engineered set of DNA substrates. Time-resolved fluorescence spectroscopy, NMR imino proton exchange measurements, and molecular dynamics simulations of the bare DNA indicated significant differences in substrate flexibilities. We observed a strong correlation between UNG efficiency and substrate flexibility, with higher kcat/KM values measured for more flexible strands. DNA bending and base flipping were observed in simulations, with more frequent uracil flipping observed for the more bendable sequences. Experiments show that bases immediately adjacent to the uracil are allosterically coupled and have the greatest impact on substrate flexibility and resultant UNG activity. A better understanding of the fundamental principles that dictate UNG activity has broad implications in diverse fields, from cancer to evolution.

How Can CpG Methylations, and Pair-to-Pair Correlations between the Main (Gene) and the Opposite Strands, Suggest a Bending DNA Loop: Insights into the 5'-UTR of DAT1.

A working hypothesis issues from patterns of methylation in the 5'-UTR of the DAT1 gene. We considered relationships between pairs of CpGs, of which one on the main-gene strand and another on the complementary opposite strand (COS). We elaborated on data from ADHD children: we calculated all possible combinations of probabilities (estimated by multiplying two raw values of methylation) in pairs of CpGs from either strand. We analyzed all correlations between any given pair and all other pairs. For pairs correlating with M6-M6COS, some pairs had cytosines positioning to the reciprocal right (e.g., M3-M2COS and M6-M5COS), other pairs had cytosines positioning to the reciprocal left (e.g., M2-M3COS; M5-M6COS). Significant pair-to-pair correlations emerged between main-strand and COS CpG pairs. Through graphic representations, we hypothesized that DNA folded to looping conformations: the C1GG C2GG C3GG and C5G C6G motifs would become close enough to allow cytosines 1-2-3 to interact with cytosines 5-6 (on both strands). Data further suggest a sliding, with left- and right-ward oscillations of DNA strands. While thorough empirical verification is needed, we hypothesize simultaneous methylation of main-strand and COS DNA ("methylation dynamics") to serve as a promising biomarker.

DeepBend: An interpretable model of DNA bendability

The bendability of genomic DNA impacts chromatin packaging and protein-DNA binding. However, we do not have a comprehensive understanding of the motifs influencing DNA bendability. Recent high-throughput technologies such as Loop-Seq offer an opportunity to address this gap but the lack of accurate and interpretable machine learning models still remains. Here we introduce DeepBend, a convolutional neural network model with convolutions designed to directly capture the motifs underlying DNA bendability and their periodic occurrences or relative arrangements that modulate bendability. DeepBend consistently performs on par with alternative models while giving an extra edge through mechanistic interpretations. Besides confirming the known motifs of DNA bendability, DeepBend also revealed several novel motifs and showed how the spatial patterns of motif occurrences influence bendability. DeepBend's genome-wide prediction of bendability further showed how bendability is linked to chromatin conformation and revealed the motifs controlling the bendability of topologically associated domains and their boundaries.

A DNA origami-based device for investigating DNA bending proteins by transmission electron microscopy.

The DNA origami technique offers precise positioning of nanoscale objects with high accuracy. This has facilitated the development of DNA origami-based functional nanomechanical devices that enable the investigation of DNA-protein interactions at the single particle level. Herein, we used the DNA origami technique to fabricate a nanoscale device for studying DNA bending proteins. For a proof of concept, we used TATA-box binding protein (TBP) to evaluate our approach. Upon binding to the TATA box, TBP causes a bend to DNA of ∼90°. Our device translates this bending into an angular change that is readily observable with a conventional transmission electron microscope (TEM). Furthermore, we investigated the roles of transcription factor II A (TF(II)A) and transcription factor II B (TF(II)B). Our results indicate that TF(II)A introduces additional bending, whereas TF(II)B does not significantly alter the TBP-DNA structure. Our approach can be readily adopted to a wide range of DNA-bending proteins and will aid the development of DNA-origami-based devices tailored for the investigation of DNA-protein interactions.

Non–B-form DNA tends to form in centromeric regions and has undergone changes in polyploid oat subgenomes

Centromeres are the specialized regions of the chromosomes that direct faithful chromosome segregation during cell division. Despite their functional conservation, centromeres display features of rapidly evolving DNA and wide evolutionary diversity in size and organization. Previous work found that the noncanonical B-form DNA structures are abundant in the centromeres of several eukaryotic species with a possible implication for centromere specification. Thus far, systematic studies into the organization and function of non-B-form DNA in plants remain scarce. Here, we applied the oat system to investigate the role of non-B-form DNA in centromeres. We conducted chromatin immunoprecipitation sequencing using an antibody to the centromere-specific histone H3 variant (CENH3); this accurately positioned oat centromeres with different ploidy levels and identified a series of centromere-specific sequences including minisatellites and retrotransposons. To define genetic characteristics of oat centromeres, we surveyed the repeat sequences and found that dyad symmetries were abundant in oat centromeres and were predicted to form non-B-DNA structures invivo. These structures including bent DNA, slipped DNA, Z-DNA, G-quadruplexes, and R-loops were prone to form within CENH3-binding regions. Dynamic conformational changes of predicted non-B-DNA occurred during the evolution from diploid to tetraploid to hexaploid oat. Furthermore, we applied the single-molecule technique of AFM and DNA:RNA immunoprecipitation with deep sequencing to validate R-loop enrichment in oat centromeres. Centromeric retrotransposons exhibited strong associations with R-loop formation. Taken together, our study elucidates the fundamental character of non-B-form DNA in the oat genome and reveals its potential role in centromeres.

Structural Plasticity of Pioneer Factor Sox2 and DNA Bendability Modulate Nucleosome Engagement and Sox2-Oct4 Synergism

Pioneer transcription factors (pTFs) can bind directly to silent chromatin and promote vital transcriptional programs. Here, by integrating high-resolution nuclear magnetic resonance (NMR) spectroscopy with biochemistry, we reveal new structural and mechanistic insights into the interaction of pluripotency pTFs and functional partners Sox2 and Oct4 with nucleosomes. We find that the affinity and conformation of Sox2 for solvent-exposed nucleosome sites depend strongly on their position and DNA sequence. Sox2, which is partially disordered but becomes structured upon DNA binding and bending, forms a super-stable nucleosome complex at superhelical location +5 (SHL+5) with similar affinity and conformation to that with naked DNA. However, at suboptimal internal and end-positioned sites where DNA may be harder to deform, Sox2 favors partially unfolded and more dynamic states that are encoded in its intrinsic flexibility. Importantly, Sox2 structure and DNA bending can be stabilized by synergistic Oct4 binding, but only on adjacent motifs near the nucleosome edge and with the full Oct4 DNA-binding domain. Further mutational studies reveal that strategically impaired Sox2 folding is coupled to reduced DNA bending and inhibits nucleosome binding and Sox2-Oct4 cooperation, while increased nucleosomal DNA flexibility enhances Sox2 association. Together, our findings fit a model where the site-specific DNA bending propensity and structural plasticity of Sox2 govern distinct modes of nucleosome engagement and modulate Sox2-Oct4 synergism. The principles outlined here can potentially guide pTF site selection in the genome and facilitate interaction with other chromatin factors or chromatin opening in vivo.

Tenuous transcriptional threshold of human sex determination. II. SRY exploits water-mediated clamp at the edge of ambiguity.

Y-encoded transcription factor SRY initiates male differentiation in therian mammals. This factor contains a high-mobility-group (HMG) box, which mediates sequence-specific DNA binding with sharp DNA bending. A companion article in this issue described sex-reversal mutations at box position 72 (residue 127 in human SRY), invariant as Tyr among mammalian orthologs. Although not contacting DNA, the aromatic ring seals the domain's minor wing at a solvent-exposed junction with a basic tail. A seeming paradox was posed by the native-like biochemical properties of inherited Swyer variant Y72F: its near-native gene-regulatory activity is consistent with the father's male development, but at odds with the daughter's XY female somatic phenotype. Surprisingly, aromatic rings (Y72, F72 or W72) confer higher transcriptional activity than do basic or polar side chains generally observed at solvated DNA interfaces (Arg, Lys, His or Gln). Whereas biophysical studies (time-resolved fluorescence resonance energy transfer and heteronuclear NMR spectroscopy) uncovered only subtle perturbations, dissociation of the Y72F complex was markedly accelerated relative to wild-type. Studies of protein-DNA solvation by molecular-dynamics (MD) simulations of an homologous high-resolution crystal structure (SOX18) suggest that Y72 para-OH anchors a network of water molecules at the tail-DNA interface, perturbed in the variant in association with nonlocal conformational fluctuations. Loss of the Y72 anchor among SRY variants presumably "unclamps" its basic tail, leading to (a) rapid DNA dissociation despite native affinity and (b) attenuated transcriptional activity at the edge of sexual ambiguity. Conservation of Y72 suggests that this water-mediated clamp operates generally among SRY and metazoan SOX domains.

Deciphering the mechanical code of the genome and epigenome.

Diverse DNA-deforming processes are impacted by the local mechanical and structural properties of DNA, which in turn depend on local sequence and epigenetic modifications. Deciphering this mechanical code (that is, this dependence) has been challenging due to the lack of high-throughput experimental methods. Here we present a comprehensive characterization of the mechanical code. Utilizing high-throughput measurements of DNA bendability via loop-seq, we quantitatively established how the occurrence and spatial distribution of dinucleotides, tetranucleotides and methylated CpG impact DNA bendability. We used our measurements to develop a physical model for the sequence and methylation dependence of DNA bendability. We validated the model by performing loop-seq on mouse genomic sequences around transcription start sites and CTCF-binding sites. We applied our model to test the predictions of all-atom molecular dynamics simulations and to demonstrate that sequence and epigenetic modifications can mechanically encode regulatory information in diverse contexts.

A nuclease-mimetic platinum nanozyme induces concurrent DNA platination and oxidative cleavage to overcome cancer drug resistance

Platinum (Pt) resistance in cancer almost inevitably occurs during clinical Pt-based chemotherapy. The spontaneous nucleotide-excision repair of cancer cells is a representative process that leads to Pt resistance, which involves the local DNA bending to facilitate the recruitment of nucleotide-excision repair proteins and subsequent elimination of Pt-DNA adducts. By exploiting the structural vulnerability of this process, we herein report a nuclease-mimetic Pt nanozyme that can target cancer cell nuclei and induce concurrent DNA platination and oxidative cleavage to overcome Pt drug resistance. We show that the Pt nanozyme, unlike cisplatin and conventional Pt nanoparticles, specifically induces the nanozyme-catalyzed cleavage of the formed Pt-DNA adducts by generating in situ reactive oxygen species, which impairs the damage recognition factors-induced DNA bending prerequisite for nucleotide-excision repair. The recruitment of downstream effectors of nucleotide-excision repair to DNA lesion sites, including xeroderma pigmentosum groups A and F, is disrupted by the Pt nanozyme in cisplatin-resistant cancer cells, allowing excessive accumulation of the Pt-DNA adducts for highly efficient cancer therapy. Our study highlights the potential benefits of applying enzymatic activities to the use of the Pt nanomedicines, providing a paradigm shift in DNA damaging chemotherapy.

Conformational Change of Transcription Factors from Search to Specific Binding: A lac Repressor Case Study.

In a process known as facilitated diffusion, DNA-binding proteins find their target sites by combining three-dimensional diffusion and one-dimensional scanning of the DNA. Following the trade-off between speed and stability, agile exploration of DNA requires loose binding, whereas, at the DNA target site, the searching protein needs to establish tight interactions with the DNA. To enable both efficient search and stable binding, DNA-binding proteins and DNA often switch conformations upon recognition. Here, we study the one-dimensional diffusion and DNA binding of the dimeric lac repressor (LacI), which was reported to adopt two different conformations when binding different conformations of DNA. Using coarse-grained molecular dynamic simulations, we studied the diffusion and the sequence-specific binding of these conformations of LacI, as well as their truncated or monomeric variants, with two DNA conformations: straight and bent. The simulations were compared to experimental observables. This study supports that linear diffusion along DNA combines tight rotation-coupled groove tracking and rotation-decoupled hopping, where the protein briefly dissociates and reassociates just a few base pairs away. Tight groove tracking is crucial for target-site recognition, while hopping speeds up the overall search process. We investigated the diffusion of different LacI conformations on DNA and show how the flexibility of LacI's hinge regions ensures agility on DNA as well as faithful groove tracking. If the hinge regions instead form α-helices at the protein-DNA interface, tight groove tracking is not possible. On the contrary, the helical hinge region is essential for tight binding to bent, specific DNA, for the formation of the specific complex. Based on our study of different encounter complexes, we argue that the conformational change in LacI and DNA bending are somewhat coupled. Our findings underline the importance of two distinct protein conformations for facilitated diffusion and specific binding, respectively.

A Simulation Study of the Effect of Naturally Occurring Point Mutations on the SRY-DNA Complex.

Molecular dynamics (MD) simulations were conducted in order to investigate the effect of the naturally occurring point mutations of the transcription factor (TF) sex-determining region Y (SRY) on the structure and dynamics of the SRY-DNA complex. The normal SRY, along with the two mutants I13T and G40R, comprising point mutations on the SRY chain, which have been clinically identified in patients with sex developmental disorders, were modeled as DNA complexes. Our modeling work aims at elucidating atomic-level structural determinants of the aberrant SRY-DNA complexation by means of μs-long MD. The results suggest that the observed disorders brought about by the G40R-DNA and I13T-DNA may arise predominantly from the destabilization of the complex being in accord with in vitro assays found elsewhere and from modifications of the DNA bending as revealed in this study. Comparative potential of mean force computations, over a sequence of short separation distances for the three complexes, verified a higher stability of the normal SRY-DNA. Examining the way the SRY mutations modulate the SRY-DNA complex dynamics at the microscopic level is important also toward elucidating molecular determinants of function for proteins capable of binding to DNA.

Atomic force microscopy measurements and model of DNA bending caused by binding of AraC protein.

Atomic force microscopy (AFM) was used to conduct single-molecule imaging of protein/DNA complexes involved in the regulation of the arabinose operon of Escherichia coli. In the presence of arabinose, the transcription regulatory protein AraC binds to a 38 bp region consisting of the araI1 and araI2 half-sites. The domain positioning of full-length AraC, when bound to DNA, was not previously known. In this study, AraC was combined with 302 and 560 bp DNA and arabinose, deposited on a mica substrate, and imaged with AFM in air. High resolution images of 560 bp DNA, where bound protein was visible, showed that AraC induces a bend in the DNA with an angle 60°± 12° with a median of 55°. These results are consistent with earlier gel electrophoresis measurements that measured the DNA bend angle based on migration rates. By using known domain structures of AraC, geometric constraints, and contacts determined from biochemical experiments, we developed a model of the tertiary and quaternary structure of DNA-bound AraC in the presence of arabinose. The DNA bend angle predicted by the model is in agreement with the measurement values. We discuss the results in view of other regulatory proteins that cause DNA bending and formation of the open complex to initiate transcription.

Cryo-EM Structure and Functional Studies of EBNA1 Binding to the Family of Repeats and Dyad Symmetry Elements of Epstein-Barr Virus oriP.

Epstein-Barr nuclear antigen 1 (EBNA1) is a multifunctional viral-encoded DNA-binding protein essential for Epstein-Barr virus (EBV) DNA replication and episome maintenance. EBNA1 binds to two functionally distinct elements at the viral origin of plasmid replication (oriP), termed the dyad symmetry (DS) element, required for replication initiation and the family of repeats (FR) required for episome maintenance. Here, we determined the cryo-electron microscopy (cryo-EM) structure of the EBNA1 DNA binding domain (DBD) from amino acids (aa) 459 to 614 and its interaction with two tandem sites at the DS and FR. We found that EBNA1 induces a strong DNA bending angle in the DS, while the FR is more linear. The N-terminal arm of the DBD (aa 444 to 468) makes extensive contact with DNA as it wraps around the minor groove, with some conformational variation among EBNA1 monomers. Mutation of variable-contact residues K460 and K461 had only minor effects on DNA binding but had abrogated oriP-dependent DNA replication. We also observed that the AT-rich intervening DNA between EBNA1 binding sites in the FR can be occupied by the EBNA1 AT hook, N-terminal domain (NTD) aa 1 to 90 to form a Zn-dependent stable complex with EBNA1 DBD on a 2×FR DNA template. We propose a model showing EBNA1 DBD and NTD cobinding at the FR and suggest that this may contribute to the oligomerization of viral episomes important for maintenance during latent infection. IMPORTANCE EBV latent infection is causally linked to diverse cancers and autoimmune disorders. EBNA1 is the viral-encoded DNA binding protein required for episomal maintenance during latent infection and is consistently expressed in all EBV tumors. The interaction of EBNA1 with different genetic elements confers different viral functions, such as replication initiation at DS and chromosome tethering at FR. Here, we used cryo-EM to determine the structure of the EBNA1 DNA-binding domain (DBD) bound to two tandem sites at the DS and at the FR. We also show that the NTD of EBNA1 can interact with the AT-rich DNA sequence between tandem EBNA1 DBD binding sites in the FR. These results provide new information on the mechanism of EBNA1 DNA binding at DS and FR and suggest a higher-order oligomeric structure of EBNA1 bound to FR. Our findings have implications for targeting EBNA1 in EBV-associated disease.

Molecular basis of anti-CRISPR operon repression by Aca10.

CRISPR-Cas systems are bacterial defense systems for fighting against invaders such as bacteriophages and mobile genetic elements. To escape destruction by these bacterial immune systems, phages have co-evolved multiple anti-CRISPR (Acr) proteins, which inhibit CRISPR-Cas function. Many acr genes form an operon with genes encoding transcriptional regulators, called anti-CRISPR-associated (Aca) proteins. Aca10 is the most recently discovered Aca family that is encoded within an operon containing acrIC7 and acrIC6 in Pseudomonas citronellolis. Here, we report the high-resolution crystal structure of an Aca10 protein to unveil the molecular basis of transcriptional repressor role of Aca10 in the acrIC7-acrIC6-aca10 operon. We identified that Aca10 forms a dimer in solution, which is critical for binding specific DNA. We also showed that Aca10 directly recognizes a 21 bp palindromic sequence in the promoter of the acr operon. Finally, we revealed that R44 of Aca10 is a critical residue involved in the DNA binding, which likely results in a high degree of DNA bending.

Tenuous Transcriptional Threshold of Human Sex Determination. I. SRY and Swyer Syndrome at the Edge of Ambiguity.

Male sex determination in mammals is initiated by SRY, a Y-encoded transcription factor. The protein contains a high-mobility-group (HMG) box mediating sequence-specific DNA bending. Mutations causing XY gonadal dysgenesis (Swyer syndrome) cluster in the box and ordinarily arise de novo. Rare inherited variants lead to male development in one genetic background (the father) but not another (his sterile XY daughter). De novo and inherited mutations occur at an invariant Tyr adjoining the motif’s basic tail (box position 72; Y127 in SRY). In SRY-responsive cell lines CH34 and LNCaP, de novo mutations Y127H and Y127C reduced SRY activity (as assessed by transcriptional activation of principal target gene Sox9) by 5- and 8-fold, respectively. Whereas Y127H impaired testis-specific enhancer assembly, Y127C caused accelerated proteasomal proteolysis; activity was in part rescued by proteasome inhibition. Inherited variant Y127F was better tolerated: its expression was unperturbed, and activity was reduced by only twofold, a threshold similar to other inherited variants. Biochemical studies of wild-type (WT) and variant HMG boxes demonstrated similar specific DNA affinities (within a twofold range), with only subtle differences in sharp DNA bending as probed by permutation gel electrophoresis and fluorescence resonance-energy transfer (FRET); thermodynamic stabilities of the free boxes were essentially identical. Such modest perturbations are within the range of species variation. Whereas our cell-based findings rationalize the de novo genotype-phenotype relationships, a molecular understanding of inherited mutation Y127F remains elusive. Our companion study uncovers cryptic biophysical perturbations suggesting that the para-OH group of Y127 anchors a novel water-mediated DNA clamp.

DNA Bending Force Facilitates Z-DNA Formation under Physiological Salt Conditions.

Z-DNA, a noncanonical helical structure of double-stranded DNA (dsDNA), plays pivotal roles in various biological processes, including transcription regulation. Mechanical stresses on dsDNA, such as twisting and stretching, help to form Z-DNA. However, the effect of DNA bending, one of the most common dsDNA deformations, on Z-DNA formation is utterly unknown. Here, we show that DNA bending induces the formation of Z-DNA, that is, more Z-DNA is formed as the bending force becomes stronger. We regulated the bending force on dsDNA by using D-shaped DNA nanostructures. The B–Z transition was observed by single-molecule fluorescence resonance energy transfer. We found that as the bending force became stronger, Z-DNA was formed at lower Mg2+ concentrations. When dsDNA contained cytosine methylations, the B–Z transition occurred at 78 mM Mg2+ (midpoint) in the absence of the bending force. However, the B–Z transition occurred at a 28-fold lower Mg2+ concentration (2.8 mM) in the presence of the bending force. Monte Carlo simulation suggested that the B–Z transition stabilizes the bent form via the formation of the B–Z junction with base extrusion, which effectively releases the bending stress on DNA. Our results clearly show that the bending force facilitates the B–Z transition under physiological salt conditions.

The integration host factor regulates multiple virulence pathways in bacterial pathogen Dickeya zeae MS2.

Dickeya zeae is an aggressive bacterial phytopathogen that infects a wide range of host plants. It has been reported that integration host factor (IHF), a nucleoid‐associated protein consisting of IHFα and IHFβ subunits, regulates gene expression by influencing nucleoid structure and DNA bending. To define the role of IHF in the pathogenesis of D. zeae MS2, we deleted either and both of the IHF subunit encoding genes ihfA and ihfB, which significantly reduced the production of cell wall‐degrading enzymes (CWDEs), an unknown novel phytotoxin and the virulence factor‐modulating (VFM) quorum‐sensing (QS) signal, cell motility, biofilm formation, and thereafter the infection ability towards both potato slices and banana seedlings. To characterize the regulatory pathways of IHF protein associated with virulence, IHF binding sites (consensus sequence 5′‐WATCAANNNNTTR‐3′) were predicted and 272 binding sites were found throughout the genome. The expression of 110 tested genes was affected by IHF. Electrophoretic mobility shift assay (EMSA) showed direct interaction of IhfA protein with the promoters of vfmE, speA, pipR, fis, slyA, prtD, hrpL, hecB, hcp, indA, hdaA, flhD, pilT, gcpJ, arcA, arcB, and lysR. This study clarified the contribution of IHF in the pathogenic process of D. zeae by controlling the production of VFM and putrescine QS signals, phytotoxin, and indigoidine, the luxR‐solo system, Fis, SlyA, and FlhD transcriptional regulators, and secretion systems from type I to type VI. Characterization of the regulatory networks of IHF in D. zeae provides a target for prevention and control of plant soft rot disease.