Minimizing Structural Heterogeneity in DNA Self-Assembled Dye Templating via DNA Origami-Tuned Conformations.

With recent advances in DNA-templated dye aggregation for leveraging and engineering molecular excitons, a need exists for minimizing structural heterogeneity. Holliday Junction complexes (HJ) are commonly used to covalently template dye aggregates on their core; however, the global conformation of HJ is detrimentally dynamic. Here, the global conformation of the HJ is selectively tuned by restricting its position and orientation by using a sheet-like DNA origami construct (DOC) physisorbed on glass. The HJ arms are fixed with four different designed interduplex angles (IDAs). Atomic force microscopy confirmed that the HJs are bound to the surface of DOC with tuned IDAs. Dye orientation distributions were determined by combining dipole imaging and super-resolution microscopy. All IDAs led to dye orientations having dispersed distributions along planes perpendicular to the HJ plane, suggesting that stacking occurred between the dye and the neighboring DNA bases. The dye-base stacking interpretation was supported by increasing the size of the core cavity. The narrowest IDA minimizes structural heterogeneity and suggests dye intercalation. A strong correlation is found between the IDA and the orientation of the dye along the HJ plane. These results show that the HJ imposes restrictions on the dye and that the dye-DNA interactions are always present regardless of global conformation. The implications of our results are discussed for the scalability of dye aggregates using DNA self-assembly. Our methodology provides an avenue for the solid-supported single-molecule characterization of molecular assemblies templated on biomolecules─such as DNA and protein templates involved in light-harvesting and catalysis─with tuned conformations and restricted in position and orientation.

Efficient DNA walker guided by ordered cruciform-shaped DNA track for ultrasensitive and rapid electrochemical detection of lead ion.

The rational design of DNA tracks is an effective pathway to guide the autonomous movement and high-efficiency recognition in DNA walkers, showing outstanding advantages for the cascade signal amplification of electrochemical biosensors. However, the uncontrolled distance between two adjacent tracks on the electrode could increase the risk of derailment and interruption of the reaction. Hence, a novel four-way balanced cruciform-shaped DNA track (C-DNT) was designed as a structured pathway to improve the effectiveness and stability of the reaction in DNA walkers. In this work, two kinds of cruciform-shaped DNA were interconnected as a robust structure that could avoid the invalid movement of the designed DNA walker on the electrode. When hairpin H2 was introduced onto the electrode, the strand displacement reaction (SDR) effectively triggered movements of the DNA walker along the cruciform-shaped track while leaving ferrocene (Fc) on the electrode, leading to a significant enhancement of the electrochemical signal. This design enabled the walker to move in an excellent organized and controllable manner, thus enhancing the reaction speed and walking efficiency. Compared to other walkers moving on random tracks, the reaction time of the C-DNT-based DNA walker could be reduced to 20 min. Lead ion (Pb2+) was used as a model target to evaluate the analytical performance of this biosensor, which exhibited a low detection limit of 0.033 pM along with a wide detection ranging from 0.1 pM to 500 nM. This strategy presented a novel concept for designing a high-performance DNA walker-based sensing platform for the detection of contaminants.

Deciphering the Substrate Specificity Reveals that CRISPR-Cas12a Is a Bifunctional Enzyme with Both Endo- and Exonuclease Activities.

The class 2 CRISPR-Cas9 and CRISPR-Cas12a systems, originally described as adaptive immune systems of bacteria and archaea, have emerged as versatile tools for genome-editing, with applications in biotechnology and medicine. However, significantly less is known about their substrate specificity, but such knowledge may provide instructive insights into their off-target cleavage and previously unrecognized mechanism of action. Here, we document that the Acidaminococcus sp. Cas12a (AsCas12a) binds preferentially, and independently of crRNA, to a suite of branched DNA structures, such as the Holliday junction (HJ), replication fork and D-loops, compared with single- or double-stranded DNA, and promotes their degradation. Further, our study revealed that AsCas12a binds to the HJ, specifically at the crossover region, protects it from DNase I cleavage and renders a pair of thymine residues in the HJ homologous core hypersensitive to KMnO4 oxidation, suggesting DNA melting and/or distortion. Notably, these structural changes enabled AsCas12a to resolve HJ into nonligatable intermediates, and subsequently their complete degradation. We further demonstrate that crRNA impedes HJ cleavage by AsCas12a, and that of Lachnospiraceae bacterium Cas12a, without affecting their DNA-binding ability. We identified a separation-of-function variant, which uncouples DNA-binding and DNA cleavage activities of AsCas12a. Importantly, we found robust evidence that AsCas12a endonuclease also has 3'-to-5' and 5'-to-3' exonuclease activity, and that these two activities synergistically promote degradation of DNA, yielding di- and mononucleotides. Collectively, this study significantly advances knowledge about the substrate specificity of AsCas12a and provides important insights into the degradation of different types of DNA substrates.

DNA digestion and formation of DNA-network structures with Holliday junction-resolving enzyme Hjc_15-6 in conjunction with polymerase reactions.

The recently identified novel Holliday junction-resolving enzyme, termed Hjc_15-6, activity investigation results imply DNA cleavage by Hjc_15-6 in a manner that potentially enhances the molecular self-assembly that may be exploited for creating DNA-networks and nanostructures. The study also demonstrates Pwo DNA polymerase acting in combination with Hjc_15-6 capability to produce large amounts of DNA that transforms into large DNA-network structures even without DNA template and primers. Furthermore, it is demonstrated that Hjc_15-6 prefers Holliday junction oligonucleotides as compared to Y-shaped oligonucleotides as well as efficiently cleaves typical branched products from isothermal DNA amplification of both linear and circular DNA templates amplified by phi29-like DNA polymerase. The assembly of large DNA network structures was observed in real time, by transmission electron microscopy, on negative stained grids that were freshly prepared, and also on the same grids after incubation for 4 days under constant cooling. Hence, Hjc_15-6 is a promising molecular tool for efficient production of various DNA origamis that may be implemented for a wide range of applications such as within medical biomaterials, catalytic materials, molecular devices and biosensors.

Ebselen and TPI-1, as RecG helicase inhibitors, potently enhance the susceptibility of Pseudomonas aeruginosa to DNA damage agents.

Holliday junction (HJ) is a four-way structured DNA intermediate in processes of homologous recombination and DNA double-stranded break (DSB) repair. In bacteria, HJs are processed via either the RuvABC or RecG-dependent pathways. In addition, RecG also plays a critical role in the reactivation of stalled replication forks, making it an attractive target for antibacterial drug development. Here, we conducted a high-throughput screening targeting the RecG helicase from a common opportunistic pathogen Pseudomonas aeruginosa (Pa). From a library containing 7920 compounds, we identified Ebselen and TPI-1 (2',5'-Dichloro-[1,1'-biphenyl]-2,5-dione) as two potent PaRecG inhibitors, with IC50 values of 0.31 ± 0.02 µM and 1.16 ± 0.06 µM, respectively. Further biochemical analyses suggested that both Ebselen and TPI-1 inhibited the ATPase activity of PaRecG, and hindered its binding to HJ DNA with high selectivity. These compounds, when combined with our previously reported RuvAB inhibitors, resulted in more severe DNA repair defects than the individual treatment, and potently enhanced the susceptibility of P. aeruginosa to the DNA damage agents. This work reports novel small molecule inhibitors of RecG, offering valuable chemical tools for advancing our understanding of RecG's function and mechanism. Additionally, these inhibitors might be further developed as promising antibacterial agents in the fight against P. aeruginosa infections.

An Albumin-Holliday Junction Biomolecular Modular Design for Programmable Multifunctionality and Prolonged Circulation.

Combinatorial properties such as long-circulation and site- and cell-specific engagement need to be built into the design of advanced drug delivery systems to maximize drug payload efficacy. This work introduces a four-stranded oligonucleotide Holliday Junction (HJ) motif bearing functional moieties covalently conjugated to recombinant human albumin (rHA) to give a "plug-and-play" rHA-HJ multifunctional biomolecular assembly with extended circulation. Electrophoretic gel-shift assays show successful functionalization and purity of the individual high-performance liquid chromatography-purified modules as well as efficient assembly of the rHA-HJ construct. Inclusion of an epidermal growth factor receptor (EGFR)-targeting nanobody module facilitates specific binding to EGFR-expressing cells resulting in approximately 150-fold increased fluorescence intensity determined by flow cytometric analysis compared to assemblies absent of nanobody inclusion. A cellular recycling assay demonstrated retained albumin-neonatal Fc receptor (FcRn) binding affinity and accompanying FcRn-driven cellular recycling. This translated to a 4-fold circulatory half-life extension (2.2 and 0.55 h, for the rHA-HJ and HJ, respectively) in a double transgenic humanized FcRn/albumin mouse. This work introduces a novel biomolecular albumin-nucleic acid construct with extended circulatory half-life and programmable multifunctionality due to its modular design.

BLM and BRCA1-BARD1 coordinate complementary mechanisms of joint DNA molecule resolution.

The Bloom syndrome helicase BLM interacts with topoisomerase IIIα (TOP3A), RMI1, and RMI2 to form the BTR complex, which dissolves double Holliday junctions and DNA replication intermediates to promote sister chromatid disjunction before cell division. In its absence, structure-specific nucleases like the SMX complex (comprising SLX1-SLX4, MUS81-EME1, and XPF-ERCC1) can cleave joint DNA molecules instead, but cells deficient in both BTR and SMX are not viable. Here, we identify a negative genetic interaction between BLM loss and deficiency in the BRCA1-BARD1 tumor suppressor complex. We show that this is due to a previously overlooked role for BARD1 in recruiting SLX4 to resolve DNA intermediates left unprocessed by BLM in the preceding interphase. Consequently, cells with defective BLM and BRCA1-BARD1 accumulate catastrophic levels of chromosome breakage and micronucleation, leading to cell death. Thus, we reveal mechanistic insights into SLX4 recruitment to DNA lesions, with potential clinical implications for treating BRCA1-deficient tumors.

Multifaceted Role of PARP1 in Maintaining Genome Stability Through Its Binding to Alternative DNA Structures.

Alternative DNA structures that differ from the canonical B-form of DNA can arise from repetitive sequences and play beneficial roles in many cellular processes such as gene regulation and chromatin organization. However, they also threaten genomic stability in several ways including mutagenesis and collisions with replication and/or transcription machinery, which lead to genomic instability that is associated with human disease. Thus, the careful regulation of non-B-DNA structure formation and resolution is crucial for the maintenance of genome integrity. Several protein factors have been demonstrated to associate with alternative DNA structures to facilitate their removal, one of which is the ADP-ribose transferase (ART) PARP1 (also called ADP-ribosyltransferase diphtheria toxin-like 1 or ARTD1), a multifaceted DNA repair enzyme that recognizes single- and double-stranded DNA breaks and synthesizes chains of poly (ADP-ribose) (PAR) to recruit DNA repair proteins. It is now well appreciated that PARP1 recognizes several nucleic acid structures beyond DNA lesions, including stalled replication forks, DNA hairpins and cruciforms, R-loops, and DNA G-quadruplexes (G4 DNA). In this review, we summarize the current evidence of a direct association of PARP1 with each of these aforementioned alternative DNA structures, as well as discuss the role of PARP1 in the prevention of non-B-DNA structure-induced genetic instability. We will focus on the mechanisms of the recognition and binding by PARP1 to each alternative structure and the structure-based stimulation of PARP1 catalytic activity upon binding. Finally, we will discuss some of the outstanding gaps in the literature and offer speculative insight for questions that remain to be experimentally addressed.

Effect of hydrophilicity-imparting substituents on exciton delocalization in squaraine dye aggregates covalently templated to DNA Holliday junctions.

Molecular aggregates exhibit emergent properties, including the collective sharing of electronic excitation energy known as exciton delocalization, that can be leveraged in applications such as quantum computing, optical information processing, and light harvesting. In a previous study, we found unexpectedly large excitonic interactions (quantified by the excitonic hopping parameter Jm,n) in DNA-templated aggregates of squaraine (SQ) dyes with hydrophilic-imparting sulfo and butylsulfo substituents. Here, we characterize DNA Holliday junction (DNA-HJ) templated aggregates of an expanded set of SQs and evaluate their optical properties in the context of structural heterogeneity. Specifically, we characterized the orientation of and Jm,n between dyes in dimer aggregates of non-chlorinated and chlorinated SQs. Three new chlorinated SQs that feature a varying number of butylsulfo substituents were synthesized and attached to a DNA-HJ via a covalent linker to form adjacent and transverse dimers. Various characteristics of the dye, including its hydrophilicity (in terms of log Po/w) and surface area, and of the substituents, including their local bulkiness and electron withdrawing capacity, were quantified computationally. The orientation of and Jm,n between the dyes were estimated using a model based on Kühn-Renger-May theory to fit the absorption and circular dichroism spectra. The results suggested that adjacent dimer aggregates of all the non-chlorinated and of the most hydrophilic chlorinated SQ dyes exhibit heterogeneity; that is, they form a mixture of dimers subpopulations. A key finding of this work is that dyes with a higher hydrophilicity (lower log Po/w) formed dimers with smaller Jm,n and large center-to-center dye distance (Rm,n). Also, the results revealed that the position of the dye in the DNA-HJ template, that is, adjacent or transverse, impacted Jm,n. Lastly, we found that Jm,n between symmetrically substituted dyes was reduced by increasing the local bulkiness of the substituent. This work provides insights into how to maintain strong excitonic coupling and identifies challenges associated with heterogeneity, which will help to improve control of these dye aggregates and move forward their potential application as quantum information systems.

Heterozygosity alters Msh5 binding to meiotic chromosomes in the baker's yeast.

Meiotic crossovers are initiated from programmed DNA double-strand breaks. The Msh4-Msh5 heterodimer is an evolutionarily conserved mismatch repair-related protein complex that promotes meiotic crossovers by stabilizing strand invasion intermediates and joint molecule structures such as Holliday junctions. In vivo studies using homozygous strains of the baker's yeast Saccharomyces cerevisiae (SK1) show that the Msh4-Msh5 complex associates with double-strand break hotspots, chromosome axes, and centromeres. Many organisms have heterozygous genomes that can affect the stability of strand invasion intermediates through heteroduplex rejection of mismatch-containing sequences. To examine Msh4-Msh5 function in a heterozygous context, we performed chromatin immunoprecipitation and sequencing (ChIP-seq) analysis in a rapidly sporulating hybrid S. cerevisiae strain (S288c-sp/YJM789, containing sporulation-enhancing QTLs from SK1), using SNP information to distinguish reads from homologous chromosomes. Overall, Msh5 localization in this hybrid strain was similar to that determined in the homozygous strain (SK1). However, relative Msh5 levels were reduced in regions of high heterozygosity, suggesting that high mismatch densities reduce levels of recombination intermediates to which Msh4-Msh5 binds. Msh5 peaks were also wider in the hybrid background compared to the homozygous strain (SK1). We determined regions containing heteroduplex DNA by detecting chimeric sequence reads with SNPs from both parents. Msh5-bound double-strand break hotspots overlap with regions that have chimeric DNA, consistent with Msh5 binding to heteroduplex-containing recombination intermediates.

Enhanced Photo-Cross-Linking of Thymines in DNA Holliday Junction-Templated Squaraine Dimers.

Programmable self-assembly of dyes using DNA templates to promote exciton delocalization in dye aggregates is gaining considerable interest. New methods to improve the rigidity of the DNA scaffold and thus the stability of the molecular dye aggregates to encourage exciton delocalization are desired. In these dye-DNA constructs, one potential way to increase the stability of the aggregates is to create an additional covalent bond via photo-cross-linking reactions between thymines in the DNA scaffold. Specifically, we report an approach to increase the yield of photo-cross-linking reaction between thymines in the core of a DNA Holliday junction while limiting the damage from UV irradiation to DNA. We investigated the effect of the distance between thymines on the photo-cross-linking reaction yields by using linkers with different lengths to tether the dyes to the DNA templates. By comprehensively evaluating the photo-cross-linking reaction yields of dye-DNA aggregates using linkers with different lengths, we conclude that interstrand thymines tend to photo-cross-link more efficiently with short linkers. A higher cross-linking yield was achieved due to the shorter intermolecular distance between thymines influenced by strong dye-dye interactions. Our method establishes the possibility of improving the stability of DNA-scaffolded dye aggregates, thereby expanding their use in exciton-based applications such as light harvesting, nanoscale computing, quantum computing, and optoelectronics.

Method to generate Holliday junction recombination intermediates via RecA-mediated four-strand exchange.

DNA molecules that contain single Holliday junctions have served as model substrates to investigate the pathway in which homologous recombination intermediates are processed. However, the preparation of DNA containing Holliday junctions in high yield remains a challenge. In this work, we used a nicking endonuclease to generate gapped DNA, from which α-structured DNA or figure-8 DNA were created via RecA-mediated reactions. The resulting DNA molecules were found to serve as good substrates for Holliday junction resolvases. The simplified method negates the requirement for radioactive labelling of DNA, making the generation of Holliday junction DNA more accessible to non-experts.

Analysis of non-canonical three- and four-way DNA junctions.

The development of compounds that can selectively bind with non-canonical DNA structures has expanded in recent years. Junction DNA, including three-way junctions (3WJs) and four-way Holliday junctions (HJs), offer an intriguing target for developmental therapeutics as both 3WJs and HJs are involved in DNA replication and repair processes. However, there are a limited number of assays available for the analysis of junction DNA binding. Here, we describe the design and execution of multiplex fluorescent polyacrylamide gel electrophoresis (PAGE) and microscale thermophoresis (MST) assays that enable evaluation of junction-binding compounds. Two well characterised junction-binding compounds-a C6 linked bis-acridine ligand and an iron(II)-bound peptide helicate, which recognise HJs and 3WJs, respectively-were employed as probes for both MST and PAGE experiments. The multiplex PAGE assay expands beyond previously reported fluorescent PAGE as it uses four individual fluorophores that can be combined to visualise single-strands, pseudo-duplexes, and junction DNA present during 3WJ and HJ formation. The use of MST to identify the binding affinity of junction binding agents is, to our knowledge, first reported example of this technique. The combined use of PAGE and MST provides complementary results for the visualisation of 3WJ and HJ formation and the direct binding affinity (Kd and EC50) of these agents. These assays can be used to aid the discovery and design of new therapeutics targeting non-canonical nucleic acid structures.

In vitro transcription of self-assembling DNA nanoparticles.

Nucleic acid nanoparticles are playing an increasingly important role in biomolecular diagnostics and therapeutics as well as a variety of other areas. The unique attributes of self-assembling DNA nanoparticles provide a potentially valuable addition or alternative to the lipid-based nanoparticles that are currently used to ferry nucleic acids in living systems. To explore this possibility, we have assessed the ability of self-assembling DNA nanoparticles to be constructed from complete gene cassettes that are capable of gene expression in vitro. In the current report, we describe the somewhat counter-intuitive result that despite extensive crossovers (the stereochemical analogs of Holliday junctions) and variations in architecture, these DNA nanoparticles are amenable to gene expression as evidenced by T7 RNA polymerase-driven transcription of a reporter gene in vitro. These findings, coupled with the vastly malleable architecture and chemistry of self-assembling DNA nanoparticles, warrant further investigation of their utility in biomedical genetics.

Holliday junction branch migration driven by AAA+ ATPase motors.

Holliday junctions are key intermediate DNA structures during genetic recombination. One of the first Holliday junction-processing protein complexes to be discovered was the well conserved RuvAB branch migration complex present in bacteria that mediates an ATP-dependent movement of the Holliday junction (branch migration). Although the RuvAB complex served as a paradigm for the processing of the Holliday junction, due to technical limitations the detailed structure and underlying mechanism of the RuvAB branch migration complex has until now remained unclear. Recently, structures of a reconstituted RuvAB complex actively-processing a Holliday junction were resolved using time-resolved cryo-electron microscopy. These structures showed distinct conformational states at different stages of the migration process. These structures made it possible to propose an integrated model for RuvAB Holliday junction branch migration. Furthermore, they revealed unexpected insights into the highly coordinated and regulated mechanisms of the nucleotide cycle powering substrate translocation in the hexameric AAA+ RuvB ATPase. Here, we review these latest advances and describe areas for future research.

Genetic dissection of crossover mutants defines discrete intermediates in mouse meiosis.

Crossovers (COs), the exchange of homolog arms, are required for accurate chromosome segregation during meiosis. Studies in yeast have described the single-end invasion (SEI) intermediate: a stabilized 3' end annealed with the homolog as the first detectible CO precursor. SEIs are thought to differentiate into double Holliday junctions (dHJs) that are resolved by MutLgamma (MLH1/MLH3) into COs. Currently, we lack knowledge of early steps of mammalian CO recombination or how intermediates are differentiated in any organism. Using comprehensive analysis of recombination in thirteen different genetic conditions with varying levels of compromised CO resolution, we infer CO precursors include asymmetric SEI-like intermediates and dHJs in mouse. In contrast to yeast, MLH3 is structurally required to differentiate CO precursors into dHJs. We verify conservation of aspects of meiotic recombination and show unique features in mouse, providing mechanistic insight into CO formation.

Molecular mechanisms of Holliday junction branch migration catalyzed by an asymmetric RuvB hexamer.

The Holliday junction (HJ) is a DNA intermediate of homologous recombination, involved in many fundamental physiological processes. RuvB, an ATPase motor protein, drives branch migration of the Holliday junction with a mechanism that had yet to be elucidated. Here we report two cryo-EM structures of RuvB, providing a comprehensive understanding of HJ branch migration. RuvB assembles into a spiral staircase, ring-like hexamer, encircling dsDNA. Four protomers of RuvB contact the DNA backbone with a translocation step size of 2 nucleotides. The variation of nucleotide-binding states in RuvB supports a sequential model for ATP hydrolysis and nucleotide recycling, which occur at separate, singular positions. RuvB's asymmetric assembly also explains the 6:4 stoichiometry between the RuvB/RuvA complex, which coordinates HJ migration in bacteria. Taken together, we provide a mechanistic understanding of HJ branch migration facilitated by RuvB, which may be universally shared by prokaryotic and eukaryotic organisms.

Organometallic Pillarplexes That Bind DNA 4-Way Holliday Junctions and Forks.

Holliday 4-way junctions are key to important biological DNA processes (insertion, recombination, and repair) and are dynamic structures that adopt either open or closed conformations, the open conformation being the biologically active form. Tetracationic metallo-supramolecular pillarplexes display aryl faces about a cylindrical core, an ideal structure to interact with open DNA junction cavities. Combining experimental studies and MD simulations, we show that an Au pillarplex can bind DNA 4-way (Holliday) junctions in their open form, a binding mode not accessed by synthetic agents before. Pillarplexes can bind 3-way junctions too, but their large size leads them to open up and expand that junction, disrupting the base pairing, which manifests in an increased hydrodynamic size and lower junction thermal stability. At high loading, they rearrange both 4-way and 3-way junctions into Y-shaped forks to increase the available junction-like binding sites. Isostructural Ag pillarplexes show similar DNA junction binding behavior but lower solution stability. This pillarplex binding contrasts with (but complements) that of metallo-supramolecular cylinders, which prefer 3-way junctions and can rearrange 4-way junctions into 3-way junction structures. The pillarplexes' ability to bind open 4-way junctions creates exciting possibilities to modulate and switch such structures in biology, as well as in synthetic nucleic acid nanostructures. In human cells, the pillarplexes do reach the nucleus, with antiproliferative activity at levels similar to those of cisplatin. The findings provide a new roadmap for targeting higher-order junction structures using a metallo-supramolecular approach, as well as expanding the toolbox available to design bioactive junction binders into organometallic chemistry.

The RuvABC Holliday Junction Processing System Is Not Required for IS26-Mediated Targeted Conservative Cointegrate Formation.

The insertion sequence IS26 plays a key role in the spread of antibiotic resistance genes in Gram-negative bacteria. IS26 and members of the IS26 family are able to use two distinct mechanisms to form cointegrates made up of two DNA molecules linked via directly oriented copies of the IS. The well-known copy-in (formerly replicative) reaction occurs at very low frequency, and the more recently discovered targeted conservative reaction, which joins two molecules that already include an IS, is substantially more efficient. Experimental evidence has indicated that, in the targeted conservative mode, the action of Tnp26, the IS26 transposase, is required only at one end. How the Holliday junction (HJ) intermediate generated by the Tnp26-catalyzed single-strand transfer is processed to form the cointegrate is not known. We recently proposed that branch migration and resolution via the RuvABC system may be needed to process the HJ; here, we have tested this hypothesis. In reactions between a wild-type and a mutant IS26, the presence of mismatched bases near one IS end impeded the use of that end. In addition, evidence of gene conversion, potentially consistent with branch migration, was detected in some of the cointegrates formed. However, the targeted conservative reaction occurred in strains that lacked the recG, ruvA, or ruvC genes. As the RuvC HJ resolvase is not required for targeted conservative cointegrate formation, the HJ intermediate formed by the action of Tnp26 must be resolved by an alternate route. IMPORTANCE In Gram-negative bacteria, the contribution of IS26 to the spread of antibiotic resistance and other genes that provide cells with an advantage under specific conditions far exceeds that of any other known insertion sequence. This is likely due to the unique mechanistic features of IS26 action, particularly its propensity to cause deletions of adjacent DNA segments and the ability of IS26 to use two distinct reaction modes for cointegrate formation. The high frequency of the unique targeted conservative reaction mode that occurs when both participating molecules include an IS26 is also key. Insights into the detailed mechanism of this reaction will help to shed light on how IS26 contributes to the diversification of the bacterial and plasmid genomes it is found in. These insights will apply more broadly to other members of the IS26 family found in Gram-positive as well as Gram-negative pathogens.

Computational analysis on the dissemination of non-B DNA structural motifs in promoter regions of 1180 cellular genomes.

The promoter regions of gene regulation are under evolutionary constraints and earlier studies uncovered that they are characterized by enrichment of functional non-B DNA structural signatures like curved DNA, cruciform DNA, G-quadruplex, triple-helical DNA, slipped DNA structures, and Z-DNA. However, these studies are restricted to a few model organisms, single non-B DNA motif types, or whole genomic sequences, and their comparative accumulation in promoter regions of different domains of life has not been reported comprehensively. In this study, for the first time, we investigated the preponderance of non-B DNA-prone motifs in promoter regions in 1180 genomes belonging to 28 taxonomic groups using the non-B DNA Motif Search Tool (nBMST). The trends suggest that they are predominant in promoters compared to the upstream and downstream regions of all three domains of life and variably linked to taxonomic groups. Cruciform DNA motif is the most abundant form of non-B DNA, spanning from archaea to lower eukaryotes. Curved DNA motifs are prominent in host-associated bacteria, and suppressed in mammals. Triplex-DNA and slipped DNA structure repeats are discretely dispersed in all lineages. G-quadruplex motifs are significantly enriched in mammals. We also observed that the unique enrichment of non-B DNA in promoters is strongly linked to genome GC, size, evolutionary time divergence, and ecological adaptations. Overall, our work systematically reports the unique non-B DNA structural landscape of cellular organisms from the perspective of the cis-regulatory code of genomes.