DNA Segments Research Articles

DNA-induces intra- and inter-molecular changes in dimerization of the human FOXP1.

Transcription factors (TFs) are proteins that regulate gene expression by binding to specific DNA sequences and gating access to genes. This paradigm implies a one-directional gene regulation of DNA remodeling via TFs and their cofactors. Here, we present an example in which the DNA modulates the kinetics of dimerization of the forkhead (FKH) domain of the FoxP1 human TF, favoring its monomeric form over its domain-swapped dimer conformation. Hence, challenging the one-dimensional remodeling paradigm. To inquire about the structural dynamics of the FKH domain in its dimeric form and the presence of DNA, we use molecular dynamics simulations, single-molecule multiparameter fluorescence spectroscopy, and biochemical assays. We found that the monomeric form of the FoxP1 FKH domain primarily displays a disordered-like behavior, which is allosterically reverted by dimerization. Interestingly, DNA favors its interaction with the FKH in its monomeric form over its domain-swapped dimer at the cost of decreasing the FKH domain structural stability. Our findings imply that gene expression is tightly regulated by a negative feedback control over suppressor proteins such as FoxP1 by introducing a bidirectional control over gene expression. Moreover, it suggests that FoxP1 gene suppression action in its domain-swapped conformation, which brings together distant DNA segments and locks transcription access, requires the presence of other dimerization domains, cofactors, or the nuclear macromolecular composition to revert the negative feedback exerted by the DNA.

Modeling and simulating human mitochondrial RNA polymerase nucleotide addition cycle using computational microscope.

Human mitochondrial RNA polymerase (POLRMT) is essential to the transcription of mitochondrial DNA and cell metabolism. This work contributes to an all-atom structural dynamics model for individual steps of the nucleotide addition cycle of POLRMT. We aim to establish a computational model using all-atom molecular dynamics to reveal Watson-Crick base pair regulation of natural nucleotide and nucleotide analog binding and insertion in POLRMT, and a model for the post-catalytic mechanochemical coupling between product release and POLRMT translocation. The models are currently constructed combining the available high-resolution structures of POLRMT in the open and closed conformations with the bound NTP and Mg2+ ions detected in structurally similar T7 bacteriophage RNA polymerase, and by additionally using curve interpolation to place a missing non-template DNA segment. The models have been evaluated for protein subdomain stability and the presence of the appropriate protein conformation, open or closed, via subdomain motion regulation on the active site. Ongoing work will establish a model for the post-translocation mechanochemical regulations that reset for a new nucleotide addition cycle. Since POLRMT is structurally similar to viral RNA polymerases, the studies are particularly important for antiviral drug design, where nucleotide analog drug candidates are expected to be well screened for POLRMT toxicity.

Explainable artificial intelligence as a reliable annotator of archaeal promoter regions

Archaea are a vast and unexplored cellular domain that thrive in a high diversity of environments, having central roles in processes mediating global carbon and nutrient fluxes. For these organisms to balance their metabolism, the appropriate regulation of their gene expression is essential. A key momentum in regulating genes responsible for the life maintenance of archaea is when transcription factor proteins bind to the promoter element. This DNA segment is conserved, which enables its exploration by machine learning techniques. Here, we trained and tested a support vector machine with 3935 known archaeal promoter sequences. All promoter sequences were coded into DNA Duplex Stability. After, we performed a model interpretation task to map the decision pattern of the classification procedure. We also used a dataset of known-promoter sequences for validation. Our results showed that an AT rich region around position − 27 upstream (relative to the start TSS) is the most conserved in the analyzed organisms. In addition, we were able to identify the BRE element (− 33), the PPE (at − 10) and a position at + 3, that provides a more understandable picture of how promoters are organized in all the archaeal organisms. Finally, we used the interpreted model to identify potential promoter sequences of 135 unannotated organisms, delivering regulatory regions annotation of archaea in a scale never accomplished before (https://pcyt.unam.mx/gene-regulation/). We consider that this approach will be useful to understand how gene regulation is achieved in other organisms apart from the already established transcription factor binding sites.

Silencing Transcription from an Influenza Reverse Genetics Plasmid in E. coli Enhances Gene Stability.

Reverse genetics (RG) systems have been instrumental for determining the molecular aspects of viral replication, pathogenesis, and for the development of therapeutics. Here, we demonstrate that genes encoding the influenza surface antigens hemagglutinin and neuraminidase have varying stability when cloned into a common RG plasmid and transformed into Escherichia coli. Using GFP as a reporter, we demonstrate that E. coli expresses the target genes in the RG plasmid at low levels. Incorporating lac operators or a transcriptional terminator into the plasmid reduced expression and stabilized the viral genes to varying degrees. Sandwiching the viral gene between two lac operators provided the largest contribution to stability and we confirmed the stabilization is Lac repressor-dependent and crucial for subsequent plasmid propagations in E. coli. Viruses rescued from the lac operator-stabilized plasmid displayed similar kinetics and titers to the original plasmid in two different viral backbones. Together, these results indicate that silencing transcription from the plasmid in E. coli helps to maintain the correct influenza gene sequence and that the lac operator addition does not impair virus production. It is envisaged that sandwiching DNA segments between lac operators can be used for reducing DNA segment instability in any plasmid that is propagated in E. coli which express the Lac repressor.

Ionic Strength and Hybridization Position near Gold Electrodes Can Significantly Improve Kinetics in DNA-Based Electrochemical Sensors.

A variety of electrochemical (EC) biosensors play critical roles in disease diagnostics. More recently, DNA-based EC sensors have been established as promising for detecting a wide range of analyte classes. Since most of these sensors rely on the high specificity of DNA hybridization for analyte binding or structural control, it is crucial to understand the kinetics of hybridization at the electrode surface. In this work, we have used methylene blue-labeled DNA strands to monitor the kinetics of DNA hybridization at the electrode surface with square-wave voltammetry. By varying the position of the double-stranded DNA segment relative to the electrode surface as well as the bulk solution's ionic strength (0.125-1.00 M), we observed significant interferences with DNA hybridization closer to the surface, with more substantial interference at lower ionic strength. As a demonstration of the effect, toehold-mediated strand displacement reactions were slowed and diminished close to the surface, while strategic placement of the DNA binding site improved reaction rates and yields. This work manifests that both the salt concentration and DNA hybridization site relative to the electrode are important factors to consider when designing DNA-based EC sensors that measure hybridization directly at the electrode surface.

A highly sensitive electrochemical biosensor via short-stranded DNA activating LAMP(H+) to regulate i-motif folds for signal transduction

Exploring loop-mediated isothermal amplification (LAMP)-based electrochemical assay for short-stranded DNA biomarkers is intriguing. Herein, a HIV-specific DNA segment (tDNA) was introduced to implement a variant LAMP reaction, where the generated by-products H+, vLAMP(H+), acted as signal transducers for developing a sensitive electrochemical biosensor. As proof-of-concept, the tDNA-recognizable complement was programmed in a template hairpin (TH) that was ended with a 3′-terminus phosphate group to avoid non-specific elongation. Upon TH simultaneously hybridizing with tDNA and a precursor hairpin (PH), the PH underwent a loop-mediated extension with Bst polymerase and dNTPs, forming a new stem-loop hairpin in another terminus. Then the elongated product containing a double stem-loop dumbbell-like DNA structure (dDDS) was displaced and released. Using dDDS as an initiator, the vLAMP reaction occurred via consecutively cycled strand displacement for self-primed DNA incorporation. The resulting tDNA-dependent vLAMP(H+) was introduced in the modified electrode surface to guide the pH-sensitive i-motif (iM) folding. Thus, a signaling hairpin labeled with electroactive ferrocene (Fc) was dehybridized and closed, thereby bringing Fc close to the electrode interface for increased current signal readout that was highly sensitive to tDNA down to 0.034 fM. Our electrochemical biosensor would be applicable for detecting various short-stranded DNA or miRNA.

Frequent copy number variants in a cohort of Mexican-Mestizo individuals

BackgroundThe human genome presents variation at distinct levels, copy number variants (CNVs) are DNA segments of variable lengths that range from several base pairs to megabases and are present at a variable number of copies in human genomes. Common CNVs have no apparent influence on the phenotype; however, some rare CNVs have been associated with phenotypic traits, depending on their size and gene content. CNVs are detected by microarrays of different densities and are generally visualized, and their frequencies analysed using the HapMap as default reference population. Nevertheless, this default reference is inadequate when the samples analysed are from people from Mexico, since population with a Hispanic genetic background are minimally represented. In this work, we describe the variation in the frequencies of four common CNVs in Mexican-Mestizo individuals.ResultsIn a cohort of 147 unrelated Mexican-Mestizo individuals, we found that the common CNVs 2p11.2 (99.6%), 8p11.22 (54.5%), 14q32.33 (100%), and 15q11.2 (71.1%) appeared with unexpectedly high frequencies when contrasted with the HapMap reference (ChAS). Yet, while when comparing to an ethnically related reference population, these differences were significantly reduced or even disappeared.ConclusionThe findings in this work contribute to (1) a better description of the CNVs characteristics of the Mexican Mestizo population and enhance the knowledge of genome variation in different ethnic groups. (2) emphasize the importance of contrasting CNVs identified in studied individuals against a reference group that—as best as possible—share the same ethnicity while keeping this relevant information in mind when conducting CNV studies at the population or clinical level.

Structure-guided mutagenesis of the capsid protein indicates that a nanovirus requires assembled viral particles for systemic infection.

Nanoviruses are plant multipartite viruses with a genome composed of six to eight circular single-stranded DNA segments. The distinct genome segments are encapsidated individually in icosahedral particles that measure ≈18 nm in diameter. Recent studies on the model species Faba bean necrotic stunt virus (FBNSV) revealed that complete sets of genomic segments rarely occur in infected plant cells and that the function encoded by a given viral segment can complement the others across neighbouring cells, presumably by translocation of the gene products through unknown molecular processes. This allows the viral genome to replicate, assemble into viral particles and infect anew, even with the distinct genome segments scattered in different cells. Here, we question the form under which the FBNSV genetic material propagates long distance within the vasculature of host plants and, in particular, whether viral particle assembly is required. Using structure-guided mutagenesis based on a 3.2 Å resolution cryogenic-electron-microscopy reconstruction of the FBNSV particles, we demonstrate that specific site-directed mutations preventing capsid formation systematically suppress FBNSV long-distance movement, and thus systemic infection of host plants, despite positive detection of the mutated coat protein when the corresponding segment is agroinfiltrated into plant leaves. These results strongly suggest that the viral genome does not propagate within the plant vascular system under the form of uncoated DNA molecules or DNA:coat-protein complexes, but rather moves long distance as assembled viral particles.

A toolkit for assembly of targeting clones for C. elegans transgenesis.

Transgenic worms are a key resource for C. elegans researchers dissecting molecular pathways using this simple metazoan model system. Transgenes provide an avenue to visualize developmental events, cellular processes as well as real-time signal events in live animals using genetically encoded sensors. Generation of these tools has become increasingly efficient with the advent of numerous integration methods including transposon, CRISPR and recombinase-mediated integration. A growing limitation in transgene production is the assembly of the targeting constructs used to direct insertion of sequences into the genome. Here we present a toolkit that facilitates rapid assembly of complex reporters using a Golden Gate (GG) cloning-based approach. Co-assembly of one to eight DNA segments into an integration vector can be routinely obtained at high efficiency using a library of entry plasmids. The toolkit consists of 20 SapI GG entry vectors and 100 SapI GG insert plasmids containing a variety of promoters, FPs, tags, linkers, ORFs, 3' UTRs and numerous components for bipartite expression systems that can be mixed to create a huge repertoire of reporter constructs. The assembly process also works well with PCR products and 5' phosphorylated double stranded oligonucleotides, and such DNAs can be used to supply novel genes, promoters, and tags into the pipeline. In addition, the toolkit also provides a series of 12 empty BsaI -based GG assembly vectors that facilitate the construction of additional SapI GG plasmids containing novel inserts. A manual outlining the entire approach is provided as an appendix as well as a Microsoft® Excel based assembly tool which allows the user to choose individual inserts among the libraries of clones at each position in the assembly template and output an annotated sequence. The assembly process can easily be multiplexed and is typically over 90% efficient. The approach is sufficiently efficient to make microinjection rather than clone generation the limiting factor in transgene generation.

The Creation of a Gene Library of Valuable Plant Species in Armenia

Using DNA banks or gene libraries is the most efficient method of protecting and managing valuable, endangered, and rare gene pools. Genomic libraries are collections of bacteria colonies with restriction sites on their genomic DNA that contain desired genes from a specific organism. The “Scientific Center of AgroBiotechnology” branch of ANAU has created gene libraries of 34 plant species, which are preserved in the National Genebank of Crops and their Wild Relatives, which forms a basis for genomic selection. This allows genetic identification, barcoding, DNA formula decoding, and, most importantly, is the basis for genome identification. Based on the results, plasmids can incorporate restriction segments of genomic DNA and be efficiently transformed. The high level of correlation between the number of necessary clones and the efficiency of transformation of all studied plant species proves the completeness of the gene pool.

Xp21 contiguous gene deletion syndrome presenting as Duchenne muscular dystrophy and glycerol kinase deficiency associated with intellectual disability: case report and review literature.

The contiguous gene deletion syndromes (CGDS) are rare genomic disorders resulting from the deletion of large segments of DNA, manifested as the concurrence of apparently unrelated clinical features. A typical example of CGDS is Xp21 contiguous gene deletion syndrome that involves GK and its neigh-boring genes (usually DMD and NR0B1) and results in a complex phenotype, which is related to the size of deletion and involved genes. Development delay and intellectual disability are almost a constant feature of patients with CGDS. We report the case of a boy with Duchenne muscular dystrophy (DMD) and glycerol kinase deficiency (GKD) as part of the contiguous gene deletion syndrome Xp2.1, in association with intellectual disability (ID) in whom multiplex ligation-dependent probe amplification (MLPA) test first identified a hemizygous deletion involving the entire dystrophin gene. Subsequently, the array CGH study identified a maternally inherited hemizygous deletion of the Xp21.2-Xp21.1 region of approximately 3.7Mb that included both DMD and GK genes confirming the diagnosis of Xp21 CGDS. Moreover, we report a review of the cases published in the literature over the last 20 years, for which a better description of the genes involved in the syndrome was available. Intellectual disability does not appear as a constant feature of the syndrome, reiterating the concept that complex GKD syndrome results from small deletions that affect closely related but separate loci for DMD, GK and adrenal hypoplasia, rather than a single large deletion including all genes. This case highlights the importance of more in-depth genetic investigations in presence of apparently unrelated clinical findings, allowing an accurate diagnosis of contiguous gene deletion syndromes.

Classical Recombinant DNA Cloning.

Traditional molecular cloning involves a series of linked experimental steps performed with the overall goal of isolating ("cloning") a specific DNA sequence-often a gene. The main purpose of cloning is to study either that DNA sequence or the RNA or protein product it encodes. Building on key enzymatic discoveries in the late 1960s, gene cloning was pioneered in the early 1970s. Since then, DNA cloning and manipulation have been used in every area of biological and biomedical research, from molecular genetics, structural biology, and developmental biology to neurobiology, ancient DNA studies, and immunology. It is a versatile technique that can be applied to a variety of starting DNA types and lengths, including cDNAs, genes, gene fragments, chromosomal regions, or shorter fragments such as PCR products and functional control regions such as enhancers or promoters. The starting DNA can originate from any cell, tissue, or organism. In this chapter we will cover traditional ("classic") molecular cloning strategy. This comprises six linked stages in which (1) PCR is used to amplify a DNA region of interest that is then (2) digested with restriction enzymes, alongside a selected vector, to produce complementary ends crucial for the two molecules to be (3) ligated by an ATP-dependent DNA ligase, creating a recombinant DNA molecule. The recombinant DNA is then (4) introduced into competent bacterial cells by transformation and (5) grown on a selective agar media, followed by (6) colony-PCR for screening purposes. We provide a worked example to demonstrate the cloning of an average-size gene (in this case the 2kb DNA ligase A gene) from E. coli into a common plasmid expression vector. We have included six color figures and two tables to depict the key stages of a classical molecular cloning protocol. If you are cloning a segment of DNA or a gene, remember that each DNA cloning experiment is unique in terms of sequence, length, and experimental purpose. However, the principles of traditional cloning covered in this chapter are the same for any DNA sequence; we have included a detailed notes section, so you should easily be able to transfer them to your own work. Some of the following chapters in this volume will cover other, more recently developed, cloning protocols.

The good, the bad and the ugly of transposable elements annotation tools.

Transposable elements are repetitive and mobile DNA segments that can be found in virtually all organisms investigated to date. Their complex structure and variable nature are particularly challenging from the genomic annotation point of view. Many softwares have been developed to automate and facilitate TEs annotation at the genomic level, but they are highly heterogeneous regarding documentation, usability and methods. In this review, we revisited the existing software for TE genomic annotation, concentrating on the most often used ones, the methodologies they apply, and usability. Building on the state of the art of TE annotation software we propose best practices and highlight the strengths and weaknesses from the available solutions.

Structure and interaction between the novel graphene-like planar biphenylene network and DNA: Molecular dynamics simulations

The study of interactions between biomolecules and nanomaterial surfaces is of great significance for the development of nanomaterials for biomedical applications. Recently, Science reported a novel graphene-like planar biphenylene network, which has attracted the attention of many researchers. In this paper, molecular dynamics simulation of DNA adsorption on the surface of biphenylene network was used to study the biocompatibility of DNA and biphenylene network. It is found by calculation that π − π stacking is the main source of DNA adsorption on the biphenylene network surface. The single-stranded DNA segment will lie flat and adsorb on the biphenyl surface. The 9 bases in single-stranded DNA are completely adsorbed to produce strong van der Waals interaction energy, resulting in severe damage to the molecular structure of ssDNA. However, double-stranded DNA molecules will be adsorbed vertically on the biphenylene network surface, and only the terminal 2 bases will be adsorbed. Meanwhile, the configuration of double-stranded DNA did not change much. In conclusion, biphenylene network has good biocompatibility to double-stranded DNA and is toxic to single-stranded DNA. It can open up the possibility of using biphenylene network in biomedicine, promote its application in the field of biomedicine in the future, and facilitate the design of DNA/biphenylene network nanomaterials. • Molecular dynamics simulations were conducted to study the adsorption process and interaction of ssDNA and dsDNA on a novel graphene-like biphenylene network (BPN) surface. • π − π stacking was the main driving force of DNA adsorption on the surface of BPN. • The ssDNA will lie flat on the surface of the BPN, but dsDNA is perpendicular to the surface of the BPN. • Most of the bases in ssDNA are adsorbed to the surface of BPN, while only the terminal bases in dsDNA are adsorbed. • SsDNA configuration will be destroyed by BPN, while dsDNA configuration is relatively well preserved.

Charge transport properties of ideal and natural DNA segments, as mutation detectors.

DNA sequences of ideal and natural geometries are examined, studying their charge transport properties as mutation detectors. Ideal means textbook geometry. Natural means naturally distorted sequences; geometry taken from available databases. A tight-binding (TB) wire model at the base-pair level is recruited, together with a transfer matrix technique. The relevant TB parameters are obtained using a linear combination of all valence orbitals of all atoms, using geometry, either ideal or natural, as the only input. The investigated DNA sequences contain: (i) point substitution mutations - specifically, the transitions guanine (G) ↔ adenine (A) - and (ii) sequences extracted from human chromosomes, modified by expanding the cytosine-adenine-guanine triplet [(CAG)n repeats] to mimic the following diseases: (a) Huntington's disease, (b) Kennedy's disease, (c) Spinocerebellar ataxia 6, (d) Spinocerebellar ataxia 7. Quantities such as eigenspectra, density of states, transmission coefficients, and the - more experimentally relevant - current-voltage (I-V) curves are studied, intending to find adequate features to recognize mutations. To this end, the normalised deviation of the I-V curve from the origin (NDIV) is also defined. The features of the NDIV seem to provide a clearer picture, being sensitive to the number of point mutations and allowing to characterise the degree of danger of developing the aforementioned diseases.

Unveiling the interdomain dynamics of type II DNA topoisomerase through all-atom simulations: Implications for understanding its catalytic cycle

Type IIA DNA topoisomerases are complex molecular nanomachines that manage topological states of the DNA molecule in the cell and play a crucial role in cellular processes such as cell division and transcription. They are also established targets of cancer chemotherapy. Starting from the available crystal structure of a fully catalytic topoisomerase IIA homodimer from Saccharomyces cerevisiae, we constructed three states of this molecular motor primarily changing the configurations of the DNA segment bound in the DNA gate and performed μs-long all-atom molecular simulations. A comprehensive analysis revealed a sliding motion within the DNA gate and a teamwork between the N-gate and DNA gate that may be associated with the necessary molecular events that allow passage of the T-segment of DNA. The observed movement of the ATPase dimer relative to the DNA domain was reflected in different interaction patterns between the K-loops of the transducer domain and the B-A-B form of the bound DNA. Based on the obtained results, we mapped simulated configurations to the structures in the proposed catalytic cycle through which type IIA topoisomerases exert their function and discussed the possible transition events. The results extend our understanding of the mechanism of action of type IIA topoisomerases and provide an atomistic interpretation of some of the observed features of these molecular motors.

The Rule of Thirds: Controlling Junction Chirality and Polarity in 3D DNA Tiles.

The successful self-assembly of tensegrity triangle DNA crystals heralded the ability to programmably construct macroscopic crystalline nanomaterials from rationally-designed, nanoscale components. This 3D DNA tile owes its "tensegrity" nature to its three rotationally stacked double helices locked together by the tensile winding of a center strand segmented into 7 base pair (bp) inter-junction regions, corresponding to two-thirds of a helical turn of DNA. All reported tensegrity triangles to date have employed turn inter-junction segments, yielding right-handed, antiparallel, "J1" junctions. Here a minimal DNA triangle motif consisting of 3-bp inter-junction segments, or one-third of a helical turn is reported. It is found that the minimal motif exhibits a reversed morphology with a left-handed tertiary structure mediated by a locally-parallel Holliday junction-the "L1" junction. This parallel junction yields a predicted helical groove matching pattern that breaks the pseudosymmetry between tile faces, and the junction morphology further suggests a folding mechanism. A Rule of Thirds by which supramolecular chirality can be programmed through inter-junction DNA segment length is identified. These results underscore the role that global topological forces play in determining local DNA architecture and ultimately point to an under-explored class of self-assembling, chiral nanomaterials for topological processes in biological systems.

An Efficient Lightweight Hybrid Model with Attention Mechanism for Enhancer Sequence Recognition

Enhancers are sequences with short motifs that exhibit high positional variability and free scattering properties. Identification of these noncoding DNA fragments and their strength are extremely important because they play a key role in controlling gene regulation on a cellular basis. The identification of enhancers is more complex than that of other factors in the genome because they are freely scattered, and their location varies widely. In recent years, bioinformatics tools have enabled significant improvement in identifying this biological difficulty. Cell line-specific screening is not possible using these existing computational methods based solely on DNA sequences. DNA segment chromatin accessibility may provide useful information about its potential function in regulation, thereby identifying regulatory elements based on its chromatin accessibility. In chromatin, the entanglement structure allows positions far apart in the sequence to encounter each other, regardless of their proximity to the gene to be acted upon. Thus, identifying enhancers and assessing their strength is difficult and time-consuming. The goal of our work was to overcome these limitations by presenting a convolutional neural network (CNN) with attention-gated recurrent units (AttGRU) based on Deep Learning. It used a CNN and one-hot coding to build models, primarily to identify enhancers and secondarily to classify their strength. To test the performance of the proposed model, parallels were drawn between enhancer-CNNAttGRU and existing state-of-the-art methods to enable comparisons. The proposed model performed the best for predicting stage one and stage two enhancer sequences, as well as their strengths, in a cross-species analysis, achieving best accuracy values of 87.39% and 84.46%, respectively. Overall, the results showed that the proposed model provided comparable results to state-of-the-art models, highlighting its usefulness.

Consistent DNA Hypomethylations in Prostate Cancer.

With approximately 1.4 million men annually diagnosed with prostate cancer (PCa) worldwide, PCa remains a dreaded threat to life and source of devastating morbidity. In recent decades, a significant decrease in age-specific PCa mortality has been achieved by increasing prostate-specific antigen (PSA) screening and improving treatments. Nevertheless, upcoming, augmented recommendations against PSA screening underline an escalating disproportion between the benefit and harm of current diagnosis/prognosis and application of radical treatment standards. Undoubtedly, new potent diagnostic and prognostic tools are urgently needed to alleviate this tensed situation. They should allow a more reliable early assessment of the upcoming threat, in order to enable applying timely adjusted and personalized therapy and monitoring. Here, we present a basic study on an epigenetic screening approach by Methylated DNA Immunoprecipitation (MeDIP). We identified genes associated with hypomethylated CpG islands in three PCa sample cohorts. By adjusting our computational biology analyses to focus on single CpG-enriched 60-nucleotide-long DNA probes, we revealed numerous consistently differential methylated DNA segments in PCa. They were associated among other genes with NOTCH3, CDK2AP1, KLK4, and ADAM15. These can be used for early discrimination, and might contribute to a new epigenetic tumor classification system of PCa. Our analysis shows that we can dissect short, differential methylated CpG-rich DNA fragments and combinations of them that are consistently present in all tumors. We name them tumor cell-specific differential methylated CpG dinucleotide signatures (TUMS).

Condensin pinches a short negatively supercoiled DNA loop during each round of ATP usage

Condensin, an SMC (structural maintenance of chromosomes) protein complex, extrudes DNA loops using an ATP‐dependent mechanism that remains to be elucidated. Here, we show how condensin activity alters the topology of the interacting DNA. High condensin concentrations restrain positive DNA supercoils. However, in experimental conditions of DNA loop extrusion, condensin restrains negative supercoils. Namely, following ATP‐mediated loading onto DNA, each condensin complex constrains a DNA linking number difference (∆Lk) of −0.4. This ∆Lk increases to −0.8 during ATP binding and resets to −0.4 upon ATP hydrolysis. These changes in DNA topology do not involve DNA unwinding, do not spread outside the condensin‐DNA complex and can occur in the absence of the condensin subunit Ycg1. These findings indicate that during ATP binding, a short DNA domain delimited by condensin is pinched into a negatively supercoiled loop. We propose that this loop is the feeding segment of DNA that is subsequently merged to enlarge an extruding loop. Such a “pinch and merge” mechanism implies that two DNA‐binding sites produce the feeding loop, while a third site, plausibly involving Ycg1, might anchor the extruding loop.