In vivo detection of DNA secondary structures using permanganate/S1 footprinting with direct adapter ligation and sequencing (PDAL-Seq).

DNA secondary structures are essential elements of the genomic landscape, playing a critical role in regulating various cellular processes. These structures refer to G-quadruplexes, cruciforms, Z-DNA or H-DNA structures, amongst others (collectively called 'non-B DNA'), which DNA molecules can adopt beyond the B conformation. DNA secondary structures have significant biological roles, and their landscape is dynamic and can rearrange due to various factors, including changes in cellular conditions, temperature, and DNA-binding proteins. Understanding this dynamic nature is crucial for unraveling their functions in cellular processes. Detecting DNA secondary structures remains a challenge. Conventional methods, such as gel electrophoresis and chemical probing, have limitations in terms of sensitivity and specificity. Emerging techniques, including next-generation sequencing and single-molecule approaches, offer promise but face challenges since these techniques are mostly limited to only one type of secondary structure. Here we describe an updated version of a technique permanganate/S1 nuclease footprinting, which uses potassium permanganate to trap single-stranded DNA regions as found in many non-B structures, in combination with S1 nuclease digest and adapter ligation to detect genome-wide non-B formation. To overcome technical hurdles, we combined this method with direct adapter ligation and sequencing (PDAL-Seq). Furthermore, we established a user-friendly pipeline available on Galaxy to standardize PDAL-Seq data analysis. This optimized method allows the analysis of many types of DNA secondary structures that form in a living cell and will advance our knowledge of their roles in health and disease.

Replication-induced DNA secondary structures drive fork uncoupling and breakage.

Sequences that form DNA secondary structures, such as G-quadruplexes (G4s) and intercalated-Motifs (iMs), are abundant in the human genome and play various physiological roles. However, they can also interfere with replication and threaten genome stability. Multiple lines of evidence suggest G4s inhibit replication, but the underlying mechanism remains unclear. Moreover, evidence of how iMs affect the replisome is lacking. Here, we reconstitute replication of physiologically derived structure-forming sequences to find that a single G4 or iM arrest DNA replication. Direct single-molecule structure detection within solid-state nanopores reveals structures form as a consequence of replication. Combined genetic and biophysical characterisation establishes that structure stability and probability of structure formation are key determinants of replisome arrest. Mechanistically, replication arrest is caused by impaired synthesis, resulting in helicase-polymerase uncoupling. Significantly, iMs also induce breakage of nascent DNA. Finally, stalled forks are only rescued by a specialised helicase, Pif1, but not Rrm3, Sgs1, Chl1 or Hrq1. Altogether, we provide a mechanism for quadruplex structure formation and resolution during replication and highlight G4s and iMs as endogenous sources of replication stress.

G-Quadruplexes as Key Transcriptional Regulators in Neglected Trypanosomatid Parasites.

G-quadruplexes (G4s) are nucleic acid secondary structures that have been linked to the functional regulation of eukaryotic organisms. G4s have been extensively characterised in humans and emerging evidence suggests that they might also be biologically relevant for human pathogens. This indicates that G4s might represent a novel class of therapeutic targets for tackling infectious diseases. Bioinformatic studies revealed a high prevalence of putative quadruplex-forming sequences (PQSs) in the genome of protozoans, which highlights their potential roles in regulating vital processes of these parasites, including DNA transcription and replication. In this work, we focus on the neglected trypanosomatid parasites, Trypanosoma and Leishmania spp., which cause debilitating and deadly diseases across the poorest populations worldwide. We review three examples where G4-formation might be key to modulate transcriptional activity in trypanosomatids, providing an overview of experimental approaches that can be used to exploit the regulatory roles and relevance of these structures to fight parasitic infections.

DeepZ: A Deep Learning Approach for Z-DNA Prediction.

Here we describe an approach that uses deep learning neural networks such as CNN and RNN to aggregate information from DNA sequence; physical, chemical, and structural properties of nucleotides; and omics data on histone modifications, methylation, chromatin accessibility, and transcription factor binding sites and data from other available NGS experiments. We explain how with the trained model one can perform whole-genome annotation of Z-DNA regions and feature importance analysis in order to define key determinants for functional Z-DNA regions.

LiveG4ID-Seq for Profiling the Dynamic Landscape of Chromatin G-Quadruplexes During Cell Cycle in Living Cells.

G-quadruplex (G4) structures exist in the single-stranded DNA of chromatin and regulate genome function. However, the native chromatin G4 landscape in living cells has yet to be fully characterized. Herein, a genetic-encoded live-cell G4 identifier probe (LiveG4ID) is constructed and its cellular localization, biocompatibility, and G4-binding specificity is evaluated. By coupling LiveG4ID with cleavage under targets and tagmentation (CUT&Tag), LiveG4ID-seq, a method for mapping native chromatin G4 landscape in living cells with high accuracy is established. Compared to the conventional G4 CUT&Tag method, LiveG4ID-seq can identify more chromatin G4 signals and have a higher ratio of true positive signals. Using LiveG4ID-seq, the dynamic landscape of chromatin G4 structures during the cell cycle is profiled. It is discovered that chromatin G4 structures are prevalent in the promoter regions of cell cycle-specific genes, even in the early M phase when the chromatin is condensed. These data demonstrate the capacity of LiveG4ID-seq to profile a more accurate G4 landscape in living cells and promote future studies on chromatin G4 structures.

S1-END-seq reveals DNA secondary structures in human cells.

DNA becomes single stranded (ssDNA) during replication, transcription, and repair. Transiently formed ssDNA segments can adopt alternative conformations, including cruciforms, triplexes, and quadruplexes. To determine whether there are stable regions of ssDNA in the human genome, we utilized S1-END-seq to convert ssDNA regions to DNA double-strand breaks, which were then processed for high-throughput sequencing. This approach revealed two predominant non-B DNA structures: cruciform DNA formed by expanded (TA)n repeats that accumulate in microsatellite unstable human cancer cell lines and DNA triplexes (H-DNA) formed by homopurine/homopyrimidine mirror repeats common across a variety of cell lines. We show that H-DNA is enriched during replication, that its genomic location is highly conserved, and that H-DNA formed by (GAA)n repeats can be disrupted by treatment with a (GAA)n-binding polyamide. Finally, we show that triplex-forming repeats are hotspots for mutagenesis. Our results identify dynamic DNA secondary structures in vivo that contribute to elevated genome instability.

Mystery of Expansion: DNA Metabolism and Unstable Repeats.

The mammalian genome mostly contains repeated sequences. Some of these repeats are in the regulatory elements of genes, and their instability, particularly the propensity to change the repeat unit number, is responsible for 36 well-known neurodegenerative human disorders. The mechanism of repeat expansion has been an unsolved question for more than 20 years. There are a few hypotheses describing models of mutation development. Every hypothesis is based on assumptions about unusual secondary structures that violate DNA metabolism processes in the cell. Some models are based on replication errors, and other models are based on mismatch repair or base excision repair errors. Additionally, it has been shown that epigenetic regulation of gene expression can influence the probability and frequency of expansion. In this review, we consider the molecular bases of repeat expansion disorders and discuss possible mechanisms of repeat expansion during cell metabolism.

Common fragile sites: protection and repair.

Common fragile sites (CFSs) are large chromosomal regions that exhibit breakage on metaphase chromosomes upon replication stress. They become preferentially unstable at the early stage of cancer development and are hotspots for chromosomal rearrangements in cancers. Increasing evidence has highlighted the complexity underlying the instability of CFSs, and a combination of multiple mechanisms is believed to cause CFS fragility. We will review recent advancements in our understanding of the molecular mechanisms underlying the maintenance of CFS stability and the relevance of CFSs to cancer-associated genome instability. We will emphasize the contribution of the structure-prone AT-rich sequences to CFS instability, which is in line with the recent genome-wide study showing that structure-forming repeat sequences are principal sites of replication stress.

Co-Localization of DNA i-Motif-Forming Sequences and 5-Hydroxymethyl-cytosines in Human Embryonic Stem Cells.

G-quadruplexes (G4s) and i-motifs (iMs) are tetraplex DNA structures. Sequences capable of forming G4/iMs are abundant near the transcription start sites (TSS) of several genes. G4/iMs affect gene expression in vitro. Depending on the gene, the presence of G4/iMs can enhance or suppress expression, making it challenging to discern the underlying mechanism by which they operate. Factors affecting G4/iM structures can provide additional insight into their mechanism of regulation. One such factor is epigenetic modification. The 5-hydroxymethylated cytosines (5hmCs) are epigenetic modifications that occur abundantly in human embryonic stem cells (hESC). The 5hmCs, like G4/iMs, are known to participate in gene regulation and are also enriched near the TSS. We investigated genomic co-localization to assess the possibility that these two elements may play an interdependent role in regulating genes in hESC. Our results indicate that amongst 15,760 G4/iM-forming locations, only 15% have 5hmCs associated with them. A detailed analysis of G4/iM-forming locations enriched in 5hmC indicates that most of these locations are in genes that are associated with cell differentiation, proliferation, apoptosis and embryogenesis. The library generated from our analysis is an important resource for investigators exploring the interdependence of these DNA features in regulating expression of selected genes in hESC.

DNA G-Quadruplexes (G4s) Modulate Epigenetic (Re)Programming and Chromatin Remodeling: Transient Genomic G4s Assist in the Establishment and Maintenance of Epigenetic Marks, While Persistent G4s May Erase Epigenetic Marks.

Here, the emerging data on DNA G-quadruplexes (G4s) as epigenetic modulators are reviewed and integrated. This concept has appeared and evolved substantially in recent years. First, persistent G4s (e.g., those stabilized by exogenous ligands) were linked to the loss of the histone code. More recently, transient G4s (i.e., those formed upon replication or transcription and unfolded rapidly by helicases) were implicated in CpG island methylation maintenance and de novo CpG methylation control. The most recent data indicate that there are direct interactions between G4s and chromatin remodeling factors. Finally, multiple findings support the indirect participation of G4s in chromatin reshaping via interactions with remodeling-related transcription factors (TFs) or damage responders. Here, the links between the above processes are analyzed; also, how further elucidation of these processes may stimulate the progress of epigenetic therapy is discussed, and the remaining open questions are highlighted.

Tissue-specific impact of stem-loops and quadruplexes on cancer breakpoints formation.

BACKGROUND: Chromosomal rearrangements are the typical phenomena in cancer genomes causing gene disruptions and fusions, corruption of regulatory elements, damage to chromosome integrity. Among the factors contributing to genomic instability are non-B DNA structures with stem-loops and quadruplexes being the most prevalent. We aimed at investigating the impact of specifically these two classes of non-B DNA structures on cancer breakpoint hotspots using machine learning approach. METHODS: We developed procedure for machine learning model building and evaluation as the considered data are extremely imbalanced and it was required to get a reliable estimate of the prediction power. We built logistic regression models predicting cancer breakpoint hotspots based on the densities of stem-loops and quadruplexes, jointly and separately. We also tested Random Forest models varying different resampling schemes (leave-one-out cross validation, train-test split, 3-fold cross-validation) and class balancing techniques (oversampling, stratification, synthetic minority oversampling). RESULTS: We performed analysis of 487,425 breakpoints from 2234 samples covering 10 cancer types available from the International Cancer Genome Consortium. We showed that distribution of breakpoint hotspots in different types of cancer are not correlated, confirming the heterogeneous nature of cancer. It appeared that stem-loop-based model best explains the blood, brain, liver, and prostate cancer breakpoint hotspot profiles while quadruplex-based model has higher performance for the bone, breast, ovary, pancreatic, and skin cancer. For the overall cancer profile and uterus cancer the joint model shows the highest performance. For particular datasets the constructed models reach high predictive power using just one predictor, and in the majority of the cases, the model built on both predictors does not increase the model performance. CONCLUSION: Despite the heterogeneity in breakpoint hotspots' distribution across different cancer types, our results demonstrate an association between cancer breakpoint hotspots and stem-loops and quadruplexes. Approximately for half of the cancer types stem-loops are the most influential factors while for the others these are quadruplexes. This fact reflects the differences in regulatory potential of stem-loops and quadruplexes at the tissue-specific level, which yet to be discovered at the genome-wide scale. The performed analysis demonstrates that influence of stem-loops and quadruplexes on breakpoint hotspots formation is tissue-specific.

The role of fork stalling and DNA structures in causing chromosome fragility.

Alternative non-B form DNA structures, also called secondary structures, can form in certain DNA sequences under conditions that produce single-stranded DNA, such as during replication, transcription, and repair. Direct links between secondary structure formation, replication fork stalling, and genomic instability have been found for many repeated DNA sequences that cause disease when they expand. Common fragile sites (CFSs) are known to be AT-rich and break under replication stress, yet the molecular basis for their fragility is still being investigated. Over the past several years, new evidence has linked both the formation of secondary structures and transcription to fork stalling and fragility of CFSs. How these two events may synergize to cause fragility and the role of nuclease cleavage at secondary structures in rare and CFSs are discussed here. We also highlight evidence for a new hypothesis that secondary structures at CFSs not only initiate fragility but also inhibit healing, resulting in their characteristic appearance.

R-loop formation during S phase is restricted by PrimPol-mediated repriming.

During DNA replication, conflicts with ongoing transcription are frequent and require careful management to avoid genetic instability. R-loops, three-stranded nucleic acid structures comprising a DNA:RNA hybrid and displaced single-stranded DNA, are important drivers of damage arising from such conflicts. How R-loops stall replication and the mechanisms that restrain their formation during S phase are incompletely understood. Here, we show in vivo how R-loop formation drives a short purine-rich repeat, (GAA)10, to become a replication impediment that engages the repriming activity of the primase-polymerase PrimPol. Further, the absence of PrimPol leads to significantly increased R-loop formation around this repeat during S phase. We extend this observation by showing that PrimPol suppresses R-loop formation in genes harbouring secondary structure-forming sequences, exemplified by G quadruplex and H-DNA motifs, across the genome in both avian and human cells. Thus, R-loops promote the creation of replication blocks at susceptible structure-forming sequences, while PrimPol-dependent repriming limits the extent of unscheduled R-loop formation at these sequences, mitigating their impact on replication.

Unusual stability exhibited by (AT)XN12(AT)Y motif associated with high fetal hemoglobin levels.

Quasi-palindromic sequences (AT)XN12(AT)Y present in HS2 (hypersensitive site 2) of the human ß-globin locus are known to be significantly associated with increased fetal hemoglobin (HbF) levels. High HbF levels in some adults arise due to pathological conditions such as sickle cell disease and ß-thalassemia. However, elevated levels of HbF are also associated with a reducing morbidity and mortality in patients with ß-thalassemia and thus ameliorate the severity of the disease. Using gel-electrophoresis, ultraviolet (UV)-thermal denaturation, and circular dichroism (CD) techniques, we demonstrated that it exhibits a hairpin-duplex equilibrium. Intramolecular species (hairpin) were observed in both low and high salt concentrations in gel assay studies displaying the unusual stability of intramolecular species even at the high counter-ion concentration. The unusual stability of hairpin secondary structures was also demonstrated by the monophasic nature of the melting profiles for the oligonucleotides which persisted at low as well as high salt and oligomer concentrations. Change in CD spectra as a function of oligomer concentration indicates that the bimolecular duplex formation is selectively favored over monomolecular hairpin formation at and above 9 µM oligomer concentration. Thus, we hypothesize that imperfect inverted repeat sequence (AT)XN12(AT)Y of HS2 of ß-globin gene LCR forms the unusually stable hairpins which may result in the formation of a cruciform structure that may be recruited for binding by various nuclear proteins that could result in elevated HbF levels. Communicated by Ramaswamy H. Sarma.

Mms1 is an assistant for regulating G-quadruplex DNA structures.

The preservation of genome stability is fundamental for every cell. Genomic integrity is constantly challenged. Among those challenges are also non-canonical nucleic acid structures. In recent years, scientists became aware of the impact of G-quadruplex (G4) structures on genome stability. It has been shown that folded G4-DNA structures cause changes in the cell, such as transcriptional up/down-regulation, replication stalling, or enhanced genome instability. Multiple helicases have been identified to regulate G4 structures and by this preserve genome stability. Interestingly, although these helicases are mostly ubiquitous expressed, they show specificity for G4 regulation in certain cellular processes (e.g., DNA replication). To this date, it is not clear how this process and target specificity of helicases are achieved. Recently, Mms1, an ubiquitin ligase complex protein, was identified as a novel G4-DNA-binding protein that supports genome stability by aiding Pif1 helicase binding to these regions. In this perspective review, we discuss the question if G4-DNA interacting proteins are fundamental for helicase function and specificity at G4-DNA structures.

DNA secondary structure formation by DNA shuffling of the conserved domains of the Cry protein of Bacillus thuringiensis.

BACKGROUND: The Cry toxins, or δ-endotoxins, are a diverse group of proteins produced by Bacillus thuringiensis. While DNA secondary structures are biologically relevant, it is unknown if such structures are formed in regions encoding conserved domains of Cry toxins under shuffling conditions. We analyzed 5 holotypes that encode Cry toxins and that grouped into 4 clusters according to their phylogenetic closeness. The mean number of DNA secondary structures that formed and the mean Gibbs free energy [Formula: see text] were determined by an in silico analysis using different experimental DNA shuffling scenarios. In terms of spontaneity, shuffling efficiency was directly proportional to the formation of secondary structures but inversely proportional to ∆G. RESULTS: The results showed a shared thermodynamic pattern for each cluster and relationships among sequences that are phylogenetically close at the protein level. The regions of the cry11Aa, Ba and Bb genes that encode domain I showed more spontaneity and thus a greater tendency to form secondary structures (<∆G). In the region of domain III; this tendency was lower (>∆G) in the cry11Ba and Bb genes. Proteins that are phylogenetically closer to Cry11Ba and Cry11Bb, such as Cry2Aa and Cry18Aa, maintained the same thermodynamic pattern. More distant proteins, such as Cry1Aa, Cry1Ab, Cry30Aa and Cry30Ca, featured different thermodynamic patterns in their DNA. CONCLUSION: These results suggest the presence of thermodynamic variations associated to the formation of secondary structures and an evolutionary relationship with regions that encode highly conserved domains in Cry proteins. The findings of this study may have a role in the in silico design of cry gene assembly by DNA shuffling techniques.

[Conformational polymorphysm of G-rich fragments of DNA Alu-repeats. II. the putative role of G-quadruplex structures in genomic rearrangements]. / Konformatsionnyi polimorfizm G-bogatykh fragmentov ALU-povtorov DNK. II. potentsial'naia rol' G-kvadrupleksnykh struktur v genomnykh perestroikakh.

Three evolutionary conserved sites of Alu repeats (PQS2, PQS3 and PQS4) were shown to form stable inter- and intramolecular G-quadruplexes (GQs) in vitro. Structures and topologies of these GQs were elucidated using spectral methods. Self-association of G-rich Alu fragments was studied. Dimeric GQ formation from two distal identical or different putative quadruplex sites - (PQS2)2, (PQS3)2 or PQS2-PQS3 - within one lengthy DNA strand was demonstrated by a FRET-based method. Oligomer PQS4 (folded into a parallel intramolecular GQ) was shown to form stacks of quadruplexes that are stabilized by stacking interactions of external G-tetrads (this was confirmed by DOSY NMR, AFM microscopy and differential CD spectroscopy). Comparative analysis of the properties of various GQs allowed us to put forward a hypothesis of two general mechanisms of intermolecular GQ-dependant genomic rearrangements: 1) formation of a dimeric GQs; 2) association of pre-folded intramolecular parallel GQs from different strands into GQ-stacks. Thus, the observed co-localization of G-rich motifs of Alu elements with double-strand break hotspots and rearrangement hotspots may be accounted for by the specific secondary structure of these motifs. At the same time, this is likely primarily due to high abundance of such G-rich Alu fragments in the genome.

[Conformational polymorphysm of G-rich fragments of DNA ALU-repeats. I. Potential noncanonical structures]. / Konformatsionnyi polimorfizm G-bogatykh fragmentov ALU-povtorov DNK. I. Nekanonicheskie struktury.

In this paper, we report results of systematic studies of conformational polymorphism of G-rich DNA fragments from Alu repeats. Alu retrotransposones are primate-specific short interspersed elements. Using the Alu sequence from the prooncogen bcl2 intron and the consensus AluSx sequence as representative examples, we determined characteristic Alu sites that are capable of adopting G-quadruplex (GQ) conformations (i.e., potential quadruplex sites - PQSAlu), and demonstrated by bioinformatics methods that those sites are Alu-specific in the human genome. Genomic frequencies of PQSAlu were assessed (~1/10000 b.p.). The sites were found to be characteristic of young (active) Alu families (Alu-Y). A recombinant DNA sequence bearing the Alu element from the human bcl2 gene (304 b.p.) and its PQS-mutant (Alu-PQS) were constructed. The formation of noncanonical structures in Alubcl2 dsDNA and the absence of such structures in the case of Alu-PQS were shown using DMS-footprinting and AFM microscopy. Expression vectors bearing wild-type and mutant Alu insertions in the promoter regions were obtained, and the effects of these insertions on the expression of the reporter gene in НЕК293 and HeLa cell lines were compared. Our findings on the spatial organization of Alu repeats may provide insight into the mechanisms of genomic rearrangements which underlie many oncological and neurodegenerative diseases.

The Complementarity of the Loop to the Stem in DNA Pseudoknots Gives Rise to Local TAT Base-Triplets.

Pseudoknots belong to an RNA structural motif that has significant roles in the biological function of RNA. An example is ribosomal frameshifting; in this mechanism, the formation of a local triplex changes the reading frame that allows for differences in the translation of mRNAs. In this work, we have used a combination of temperature-dependent UV spectroscopy and differential scanning calorimetry (DSC) to determine the unfolding thermodynamics of a set of DNA pseudoknots with the following sequence: d(TCTCTTnAAAAAAAAGAGAT5TTTTTTT), where "Tn" is a thymine loop with n=5 (PsK-5), 7 (PsK-7), 9 (PsK-9), or 11 (PsK-11). All four oligonucleotides form intramolecular pseudoknots, and the increase in the length of this loop yielded more stable pseudoknots due to higher transition temperatures and higher unfolding enthalpies. This indicates formation of one and three TAT/TAT stacks in PsK-9 and PsK-11, respectively. We have flipped one AT for a TA base pair in the core stem of these pseudoknots, preventing in this way the formation of these base-triplet stacks. The DSC curves of these pseudoknots yielded lower unfolding enthalpies, confirming the formation of a local triplex in PsK-9 and PsK-11. Furthermore, we have investigated the reaction of PsK-5 and PsK-9 with their partially complementary strands: directly by isothermal titration calorimetry and indirectly by creating a Hess cycle with the DSC data. Relative to the PsK-5 reaction, PsK-9 reacts with its complementary strand with less favorable free energy and enthalpy contributions; this indicates PsK-9 is more stable and more compact due to the formation of a local triplex.