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Local variation of DNA structure and its dynamic nature play an essential role in the regulation of important biological processes. One of the most prominent noncanonical structures are G-quadruplexes, which form in vivo within guanine-rich regions and have been demonstrated to be involved in the regulation of transcription, translation and telomere maintenance. We provide an analysis of G-quadruplex formation in sequences with five and six guanine residues long G-tracts, which have emerged from the investigation of the gapless human genome and are associated with genes related to cancer and neurodegenerative diseases. We systematically explored the effect of G-tract and loop elongations by means of NMR and CD spectroscopy and polyacrylamide electrophoresis. Despite both types of elongation leading up to structural polymorphism, we successfully determined the topologies of four out of eight examined sequences, one of which contributes to a very scarce selection of currently known intramolecular four G-quartet structures in potassium solutions. We demonstrate that examined sequences are incompatible with five or six G-quartet structures with propeller loops, although the compatibility with other loop types cannot be factored out. Lastly, we propose a novel approach towards specific G-quadruplex targeting that could be implemented in structures with more than four G-quartets., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Current methods of processing archaeological samples combined with advances in sequencing methods lead to disclosure of a large part of H. neanderthalensis and Denisovans genetic information. It is hardly surprising that the genome variability between modern humans, Denisovans and H. neanderthalensis is relatively limited. Genomic studies may provide insight on the metabolism of extinct human species or lineages. Detailed analysis of G-quadruplex sequences in H. neanderthalensis and Denisovans mitochondrial DNA showed us interesting features. Relatively similar patterns in mitochondrial DNA are found compared to modern humans, with one notable exception for H. neanderthalensis . An interesting difference between H. neanderthalensis and H. sapiens corresponds to a motif found in the D-loop region of mtDNA, which is responsible for mitochondrial DNA replication. This area is directly responsible for the number of mitochondria and consequently for the efficient energy metabolism of cell . H. neanderthalensis harbor a long uninterrupted run of guanines in this region, which may cause problems for replication, in contrast with H. sapiens , for which this run is generally shorter and interrupted. One may propose that the predominant H. sapiens motif provided a selective advantage for modern humans regarding mtDNA replication and function., (© The Author(s) 2024. Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics.)

Nucleic acids are not only static carriers of genetic information but also play vital roles in controlling cellular lifecycles through their fascinating structural diversity [...].

Hepatitis delta virus (HDV) is a highly unusual RNA satellite virus that depends on the presence of hepatitis B virus (HBV) to be infectious. Its compact and variable single-stranded RNA genome consists of eight major genotypes distributed unevenly across different continents. The significance of noncanonical secondary structures such as G-quadruplexes (G4s) is increasingly recognized at the DNA and RNA levels, particularly for transcription, replication, and translation. G4s are formed from guanine-rich sequences and have been identified in the vast majority of viral, eukaryotic, and prokaryotic genomes. In this study, we analyzed the G4 propensity of HDV genomes by using G4Hunter. Unlike HBV, which has a G4 density similar to that of the human genome, HDV displays a significantly higher number of potential quadruplex-forming sequences (PQS), with a density more than four times greater than that of the human genome. This finding suggests a critical role for G4s in HDV, especially given that the PQS regions are conserved across HDV genotypes. Furthermore, the prevalence of G4-forming sequences may represent a promising target for therapeutic interventions to control HDV replication., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

The effect of mutations in the P53 family of transcription factors on their biological functions, including partial or complete loss of transcriptional activity, has been confirmed several times. At present, P53 family proteins showing partial loss of activity appear to be promising potential candidates for the development of novel therapeutic strategies which could restore their transcriptional activity. In this context, it is important to employ tools to precisely monitor their activity; in relation to this, non-canonical DNA secondary structures in promoters including G-quadruplexes (G4s) were shown to influence the activity of transcription factors. Here, we used a defined yeast assay to evaluate the impact of differently modeled G4 forming sequences on a panel of partial function P53 family mutant proteins. Specifically, a 22-mer G4 prone sequence (derived from the KSHV virus) and five derivatives that progressively mutate characteristic guanine stretches were placed upstream of a minimal promoter, adjacent to a P53 response element in otherwise isogenic yeast luciferase reporter strains. The transactivation ability of cancer-associated P53 (TA-P53α: A161T, R213L, N235S, V272L, R282W, R283C, R337C, R337H, and G360V) or Ectodermal Dyplasia syndromes-related P63 mutant proteins (ΔN-P63α: G134D, G134V and inR155) were tested. Our results show that the presence of G4 forming sequences can increase the transactivation ability of partial function P53 family proteins. These observations are pointing to the importance of DNA structural characteristics for accurate classification of P53 family proteins functionality in the context of the wide variety of TP53 and TP63 germline and somatic mutations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Non-canonical secondary structures in DNA are increasingly being revealed as critical players in DNA metabolism, including modulating the accessibility and activity of promoters. These structures comprise the so-called G-quadruplexes (G4s) that are formed from sequences rich in guanine bases. Using a well-defined transcriptional reporter system, we sought to systematically investigate the impact of the presence of G4 structures on transcription in yeast Saccharomyces cerevisiae. To this aim, different G4 prone sequences were modeled to vary the chance of intramolecular G4 formation, analyzed in vitro by Thioflavin T binding test and circular dichroism and then placed at the yeast ADE2 locus on chromosome XV, downstream and adjacent to a P53 response element (RE) and upstream from a minimal CYC1 promoter and Luciferase 1 (LUC1) reporter gene in isogenic strains. While the minimal CYC1 promoter provides basal reporter activity, the P53 RE enables LUC1 transactivation under the control of P53 family proteins expressed under the inducible GAL1 promoter. Thus, the impact of the different G4 prone sequences on both basal and P53 family protein-dependent expression was measured after shifting cells onto galactose containing medium. The results showed that the presence of G4 prone sequences upstream of a yeast minimal promoter increased its basal activity proportionally to their potential to form intramolecular G4 structures; consequently, this feature, when present near the target binding site of P53 family transcription factors, can be exploited to regulate the transcriptional activity of P53, P63 and P73 proteins., (© 2023 The Author(s).)

The interaction between proteins and nucleic acids is a core element of life. Many proteins bind nucleic acids via a sequence-specific manner, but there are also many types of proteins that recognize various structural motifs. Researchers have recently found that proteins that can recognize DNA and RNA G-quadruplexes (G4s) are very important for basic cellular processes, particularly in eukaryotes. Some of these proteins are located outside the nucleus and interact with RNA, potentially affecting miRNA functions in intercellular communication, which is facilitated by small extracellular vesicles (sEVs). Imbalances in the production of sEVs are associated with various pathologies and senescence in humans. The distribution of miRNA into sEVs is regulated by two RNA-binding proteins, Alyref and FUS. Both proteins possess G-rich recognition motifs that are compatible with the formation of RNA parallel G4 structures. This lends credence to the new hypothesis that G4-formation in RNAs and their interaction with G4-binding proteins can affect the fate of miRNAs and control their distribution in sEVs that are associated with senescence and aging., Competing Interests: Declaration of competing interest None declared., (Copyright © 2023 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Noncanonical secondary structures in nucleic acids have been studied intensively in recent years. Important biological roles of cruciform structures formed by inverted repeats (IRs) have been demonstrated in diverse organisms, including humans. Using Palindrome analyser, we analyzed IRs in all accessible bacterial genome sequences to determine their frequencies, lengths, and localizations. IR sequences were identified in all species, but their frequencies differed significantly across various evolutionary groups. We detected 242,373,717 IRs in all 1,565 bacterial genomes. The highest mean IR frequency was detected in the Tenericutes (61.89 IRs/kbp) and the lowest mean frequency was found in the Alphaproteobacteria (27.08 IRs/kbp). IRs were abundant near genes and around regulatory, tRNA, transfer-messenger RNA (tmRNA), and rRNA regions, pointing to the importance of IRs in such basic cellular processes as genome maintenance, DNA replication, and transcription. Moreover, we found that organisms with high IR frequencies were more likely to be endosymbiotic, antibiotic producing, or pathogenic. On the other hand, those with low IR frequencies were far more likely to be thermophilic. This first comprehensive analysis of IRs in all available bacterial genomes demonstrates their genomic ubiquity, nonrandom distribution, and enrichment in genomic regulatory regions. IMPORTANCE Our manuscript reports for the first time a complete analysis of inverted repeats in all fully sequenced bacterial genomes. Thanks to the availability of unique computational resources, we were able to statistically evaluate the presence and localization of these important regulatory sequences in bacterial genomes. This work revealed a strong abundance of these sequences in regulatory regions and provides researchers with a valuable tool for their manipulation., Competing Interests: The authors declare no conflict of interest.

Hepatitis B virus (HBV) is one of the most dangerous human pathogenic viruses found in all corners of the world. Recent sequencing of ancient HBV viruses revealed that these viruses have accompanied humanity for several millenia. As G-quadruplexes are considered to be potential therapeutic targets in virology, we examined G-quadruplex-forming sequences (PQS) in modern and ancient HBV genomes. Our analyses showed the presence of PQS in all 232 tested HBV genomes, with a total number of 1258 motifs and an average frequency of 1.69 PQS per kbp. Notably, the PQS with the highest G4Hunter score in the reference genome is the most highly conserved. Interestingly, the density of PQS motifs is lower in ancient HBV genomes than in their modern counterparts (1.5 and 1.9/kb, respectively). This modern frequency of 1.90 is very close to the PQS frequency of the human genome (1.93) using identical parameters. This indicates that the PQS content in HBV increased over time to become closer to the PQS frequency in the human genome. No statistically significant differences were found between PQS densities in HBV lineages found in different continents. These results, which constitute the first paleogenomics analysis of G4 propensity, are in agreement with our hypothesis that, for viruses causing chronic infections, their PQS frequencies tend to converge evolutionarily with those of their hosts, as a kind of 'genetic camouflage' to both hijack host cell transcriptional regulatory systems and to avoid recognition as foreign material., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

IFI16 (Interferon inducible protein 16) is a DNA sensor responsible for innate immune response stimulation and a direct viral restriction by modulating gene expression and replication. Many IFI16-DNA binding properties were described - length-dependent and sequence-independent binding, oligomerization of IFI16 upon recognition, sliding on the DNA, and preference for supercoiled DNA. However, the question of the role of IFI16-DNA binding in distinct IFI16 functions remains unclear. Here we demonstrate two modes of IFI16 binding to DNA using atomic force microscopy and electrophoretic mobility shift assays. In our study, we show that IFI16 can bind to DNA in the form of globular complexes or oligomers depending on DNA topology and molar ratios. The stability of the complexes is different in higher salt concentrations. In addition, we observed no preferential binding with the HIN-A or HIN-B domains to supercoiled DNA, revealing the importance of the whole protein for this specificity. These results provide more profound insight into IFI16-DNA interactions and may be important in answering the question of self- and non-self-DNA binding by the IFI16 protein and potentially could shed light on the role of DNA binding in distinct IFI16 functions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Guanine-quadruplex structures (G4) are unusual nucleic acid conformations formed by guanine-rich DNA and RNA sequences and known to control gene expression mechanisms, from transcription to protein synthesis. So far, a number of molecules that recognize G4 have been developed for potential therapeutic applications in human pathologies, including cancer and infectious diseases. These molecules are called G4 ligands. When the biological effects of G4 ligands are studied, the analysis is often limited to nucleic acid targets. However, recent evidence indicates that G4 ligands may target other cellular components and compartments such as lysosomes and mitochondria. Here, we summarize our current knowledge of the regulation of lysosome by G4 ligands, underlying their potential functional impact on lysosome biology and autophagic flux, as well as on the transcriptional regulation of lysosomal genes. We outline the consequences of these effects on cell fate decisions and we systematically analyzed G4-prone sequences within the promoter of 435 lysosome-related genes. Finally, we propose some hypotheses about the mechanisms involved in the regulation of lysosomes by G4 ligands.

The p53 protein is a key tumor suppressor and the most commonly mutated and down-regulated protein in human tumors. It functions mainly through interaction with DNA, and p53 acts as a transcription factor that recognizes the so-called p53 target sites on the promoters of various genes. P53 has been shown to exist as many isoforms, including three C-terminal isoforms that are produced by alternative splicing. Because the C-terminal domain is responsible for sequence-nonspecific binding and regulation of p53 binding, we have analyzed DNA recognition by these C-terminal isoforms. Using atomic force microscopy, we show for the first time that all C-terminal isoforms recognize superhelical DNA. It is particularly noteworthy that a sequence-specific p53 consensus binding site is bound by p53α and β isoforms with similar affinities, whilst p53α shows higher binding to a quadruplex sequence than both p53β and p53γ, and p53γ loses preferential binding to both the consensus binding sequence and the quadruplex-forming sequence. These results show the important role of the variable p53 C-terminal amino acid sequences for DNA recognition., (Copyright © 2022 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Sequences of nucleic acids with the potential to form four-stranded G-quadruplex structures are intensively studied mainly in the context of human diseases, pathogens, or extremophile organisms; nonetheless, the knowledge about their occurrence and putative role in plants is still limited. This work is focused on G-quadruplex-forming sites in two gene sets of interest: drought stress-responsive genes, and genes related to the production/biosynthesis of phenolic compounds in the model plant organism Arabidopsis thaliana . In addition, 20 housekeeping genes were analyzed as well, where the constitutive gene expression was expected (with no need for precise regulation depending on internal or external factors). The results have shown that none of the tested gene sets differed significantly in the content of G-quadruplex-forming sites, however, the highest frequency of G-quadruplex-forming sites was found in the 5'-UTR regions of phenolic compounds' biosynthesis genes, which indicates the possibility of their regulation at the mRNA level. In addition, mainly within the introns and 1000 bp flanks downstream gene regions, G-quadruplex-forming sites were highly underrepresented. Finally, cluster analysis allowed us to observe similarities between particular genes in terms of their PQS characteristics. We believe that the original approach used in this study may become useful for further and more comprehensive bioinformatic studies in the field of G-quadruplex genomics.

Non-B nucleic acids structures have arisen as key contributors to genetic variation in SARS-CoV-2. Herein, we investigated the presence of defining spike protein mutations falling within inverted repeats (IRs) for 18 SARS-CoV-2 variants, discussed the potential roles of G-quadruplexes (G4s) in SARS-CoV-2 biology, and identified potential pseudoknots within the SARS-CoV-2 genome. Surprisingly, there was a large variation in the number of defining spike protein mutations arising within IRs between variants and these were more likely to occur in the stem region of the predicted hairpin stem-loop secondary structure. Notably, mutations implicated in ACE2 binding and propagation (e.g., ΔH69/V70, N501Y, and D614G) were likely to occur within IRs, whilst mutations involved in antibody neutralization and reduced vaccine efficacy (e.g., T19R, ΔE156, ΔF157, R158G, and G446S) were rarely found within IRs. We also predicted that RNA pseudoknots could predominantly be found within, or next to, 29 mutations found in the SARS-CoV-2 spike protein. Finally, the Omicron variants BA.2, BA.4, BA.5, BA.2.12.1, and BA.2.75 appear to have lost two of the predicted G4-forming sequences found in other variants. These were found in nsp2 and the sequence complementary to the conserved stem-loop II-like motif (S2M) in the 3' untranslated region (UTR). Taken together, non-B nucleic acids structures likely play an integral role in SARS-CoV-2 evolution and genetic diversity.

The current monkeypox virus (MPXV) strain differs from the strain arising in 2018 by 50+ single nucleotide polymorphisms (SNPs) and is mutating much faster than expected. The cytidine deaminase apolipoprotein B messenger RNA editing enzyme, catalytic subunit B (APOBEC3) was hypothesized to be driving this increased mutation. APOBEC has recently been identified to preferentially mutate cruciform DNA secondary structures formed by inverted repeats (IRs). IRs were recently identified as hot spots for mutation in severe acute respiratory syndrome coronavirus 2, and we aimed to identify whether IRs were also hot spots for mutation within MPXV genomes. We found that MPXV genomes were replete with IR sequences. Of the 50+ SNPs identified in the 2022 outbreak strain, 63.9% of these were found to have arisen within IR regions in the 2018 reference strain (MT903344.1). Notably, IR sequences found in the 2018 reference strain were significantly lost over time, with an average of 32.5% of these sequences being conserved in the 2022 MPXV genomes. This evidence was highly indicative that mutations were arising within IRs. This data provides further support to the hypothesis that APOBEC may be driving MPXV mutation and highlights the necessity for greater surveillance of IRs of MPXV genomes to detect new mutations., (© 2022 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

G-quadruplexes (G4s) have been long considered rare and physiologically unimportant in vitro curiosities, but recent methodological advances have proved their presence and functions in vivo. Moreover, in addition to their functional relevance in bacteria and animals, including humans, their importance has been recently demonstrated in evolutionarily distinct plant species. In this study, we analyzed the genome of Pisum sativum (garden pea, or the so-called green pea), a unique member of the Fabaceae family. Our results showed that this genome contained putative G4 sequences (PQSs). Interestingly, these PQSs were located nonrandomly in the nuclear genome. We also found PQSs in mitochondrial (mt) and chloroplast (cp) DNA, and we experimentally confirmed G4 formation for sequences found in these two organelles. The frequency of PQSs for nuclear DNA was 0.42 PQSs per thousand base pairs (kbp), in the same range as for cpDNA (0.53/kbp), but significantly lower than what was found for mitochondrial DNA (1.58/kbp). In the nuclear genome, PQSs were mainly associated with regulatory regions, including 5'UTRs, and upstream of the rRNA region. In contrast to genomic DNA, PQSs were located around RNA genes in cpDNA and mtDNA. Interestingly, PQSs were also associated with specific transposable elements such as TIR and LTR and around them, pointing to their role in their spreading in nuclear DNA. The nonrandom localization of PQSs uncovered their evolutionary and functional significance in the Pisum sativum genome.

Cruciforms occur when inverted repeat sequences in double-stranded DNA adopt intra-strand hairpins on opposing strands. Biophysical and molecular studies of these structures confirm their characterization as four-way junctions and have demonstrated that several factors influence their stability, including overall chromatin structure and DNA supercoiling. Here, we review our understanding of processes that influence the formation and stability of cruciforms in genomes, covering the range of sequences shown to have biological significance. It is challenging to accurately sequence repetitive DNA sequences, but recent advances in sequencing methods have deepened understanding about the amounts of inverted repeats in genomes from all forms of life. We highlight that, in the majority of genomes, inverted repeats are present in higher numbers than is expected from a random occurrence. It is, therefore, becoming clear that inverted repeats play important roles in regulating many aspects of DNA metabolism, including replication, gene expression, and recombination. Cruciforms are targets for many architectural and regulatory proteins, including topoisomerases, p53, Rif1, and others. Notably, some of these proteins can induce the formation of cruciform structures when they bind to DNA. Inverted repeat sequences also influence the evolution of genomes, and growing evidence highlights their significance in several human diseases, suggesting that the inverted repeat sequences and/or DNA cruciforms could be useful therapeutic targets in some cases.

SARS-CoV-2 is a novel positive-sense single-stranded RNA virus from the Coronaviridae family (genus Betacoronavirus), which has been established as causing the COVID-19 pandemic. The genome of SARS-CoV-2 is one of the largest among known RNA viruses, comprising of at least 26 known protein-coding loci. Studies thus far have outlined the coding capacity of the positive-sense strand of the SARS-CoV-2 genome, which can be used directly for protein translation. However, it has been recently shown that transcribed negative-sense viral RNA intermediates that arise during viral genome replication from positive-sense viruses can also code for proteins. No studies have yet explored the potential for negative-sense SARS-CoV-2 RNA intermediates to contain protein-coding loci. Thus, using sequence and structure-based bioinformatics methodologies, we have investigated the presence and validity of putative negative-sense ORFs (nsORFs) in the SARS-CoV-2 genome. Nine nsORFs were discovered to contain strong eukaryotic translation initiation signals and high codon adaptability scores, and several of the nsORFs were predicted to interact with RNA-binding proteins. Evolutionary conservation analyses indicated that some of the nsORFs are deeply conserved among related coronaviruses. Three-dimensional protein modeling revealed the presence of higher order folding among all putative SARS-CoV-2 nsORFs, and subsequent structural mimicry analyses suggest similarity of the nsORFs to DNA/RNA-binding proteins and proteins involved in immune signaling pathways. Altogether, these results suggest the potential existence of still undescribed SARS-CoV-2 proteins, which may play an important role in the viral lifecycle and COVID-19 pathogenesis., (© The Author(s) 2022. Published by Oxford University Press.)

Parasitic helminths infecting humans are highly prevalent infecting ∼2 billion people worldwide, causing inflammatory responses, malnutrition and anemia that are the primary cause of morbidity. In addition, helminth infections of cattle have a significant economic impact on livestock production, milk yield and fertility. The etiological agents of helminth infections are mainly Nematodes (roundworms) and Platyhelminths (flatworms). G-quadruplexes (G4) are unusual nucleic acid structures formed by G-rich sequences that can be recognized by specific G4 ligands. Here we used the G4Hunter Web Tool to identify and compare potential G4 sequences (PQS) in the nuclear and mitochondrial genomes of various helminths to identify G4 ligand targets. PQS are nonrandomly distributed in these genomes and often located in the proximity of genes. Unexpectedly, a Nematode, Ascaris lumbricoides, was found to be highly enriched in stable PQS. This species can tolerate high-stability G4 structures, which are not counter selected at all, in stark contrast to most other species. We experimentally confirmed G4 formation for sequences found in four different parasitic helminths. Small molecules able to selectively recognize G4 were found to bind to Schistosoma mansoni G4 motifs. Two of these ligands demonstrated potent activity both against larval and adult stages of this parasite., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

G-quadruplexes have important regulatory roles in the nuclear genome but their distribution and potential roles in mitochondrial DNA (mtDNA) are poorly understood. We analysed 11883 mtDNA sequences from 18 taxonomic sub-groups and identified their frequency and location within mtDNA. Large differences in both the frequency and number of putative quadruplex-forming sequences (PQS) were observed amongst all the organisms and PQS frequency was negatively correlated with an increase in evolutionary age. PQS were over-represented in the 3'UTRs, D-loops, replication origins, and stem loops, indicating regulatory roles for quadruplexes in mtDNA. Variations of the G-quadruplex-forming sequence in the conserved sequence block II (CSBII) region of the human D-loop were conserved amongst other mammals, amphibians, birds, reptiles, and fishes. This D-loop PQS was conserved in the duplicated control regions of some birds and reptiles, indicating its importance to mitochondrial function. The guanine tracts in these PQS also displayed significant length heterogeneity and the length of these guanine tracts were generally longest in bird mtDNA. This information provides further insights into how G4s may contribute to the regulation and function of mtDNA and acts as a database of information for future studies investigating mitochondrial G4s in organisms other than humans., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Taking advantage of evolving and improving sequencing methods, human chromosome 8 is now available as a gapless, end-to-end assembly. Thanks to advances in long-read sequencing technologies, its centromere, telomeres, duplicated gene families and repeat-rich regions are now fully sequenced. We were interested to assess if the new assembly altered our understanding of the potential impact of non-B DNA structures within this completed chromosome sequence. It has been shown that non-B secondary structures, such as G-quadruplexes, hairpins and cruciforms, have important regulatory functions and potential as targeted therapeutics. Therefore, we analysed the presence of putative G-quadruplex forming sequences and inverted repeats in the current human reference genome (GRCh38) and in the new end-to-end assembly of chromosome 8. The comparison revealed that the new assembly contains significantly more inverted repeats and G-quadruplex forming sequences compared to the current reference sequence. This observation can be explained by improved accuracy of the new sequencing methods, particularly in regions that contain extensive repeats of bases, as is preferred by many non-B DNA structures. These results show a significant underestimation of the prevalence of non-B DNA secondary structure in previous assembly versions of the human genome and point to their importance being not fully appreciated. We anticipate that similar observations will occur as the improved sequencing technologies fill in gaps across the genomes of humans and other organisms., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Z-DNA and Z-RNA are functionally important left-handed structures of nucleic acids, which play a significant role in several molecular and biological processes including DNA replication, gene expression regulation and viral nucleic acid sensing. Most proteins that have been proven to interact with Z-DNA/Z-RNA contain the so-called Zα domain, which is structurally well conserved. To date, only eight proteins with Zα domain have been described within a few organisms (including human, mouse, Danio rerio , Trypanosoma brucei and some viruses). Therefore, this paper aimed to search for new Z-DNA/Z-RNA binding proteins in the complete PDB structures database and from the AlphaFold2 protein models. A structure-based similarity search found 14 proteins with highly similar Zα domain structure in experimentally-defined proteins and 185 proteins with a putative Zα domain using the AlphaFold2 models. Structure-based alignment and molecular docking confirmed high functional conservation of amino acids involved in Z-DNA/Z-RNA, suggesting that Z-DNA/Z-RNA recognition may play an important role in a variety of cellular processes.

G-quadruplexes are non-B secondary structures with regulatory functions and therapeutic potential. Improvements in sequencing methods recently allowed the completion of the first human chromosome which is now available as a gapless, end-to-end assembly, with the previously remaining spaces filled and newly identified regions added. We compared the presence of G-quadruplex forming sequences in the current human reference genome (GRCh38) and in the new end-to-end assembly of the X chromosome constructed by high-coverage ultra-long-read nanopore sequencing. This comparison revealed that, even though the corrected length of the chromosome X assembly is surprisingly 1.14% shorter than expected, the number of G-quadruplex forming sequences found in this gapless chromosome is significantly higher, with 493 new motifs having G4Hunter scores above 1.4 and 23 new sequences with G4Hunter scores above 3.5. This observation reflects an improved precision of the new sequencing approaches and points to an underestimation of G-quadruplex propensity in the previous, widely used version of the human genome assembly, especially for motifs with a high G4Hunter score, expected to be very stable. These G-quadruplex forming sequences probably remained undiscovered in earlier genome datasets due to previously unsolved G-rich and repetitive genomic regions. These observations allow a precise targeting of these important regulatory regions., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

R-loops are common non-B nucleic acid structures formed by a three-stranded nucleic acid composed of an RNA-DNA hybrid and a displaced single-stranded DNA (ssDNA) loop. Because the aberrant R-loop formation leads to increased mutagenesis, hyper-recombination, rearrangements, and transcription-replication collisions, it is regarded as important in human diseases. Therefore, its prevalence and distribution in genomes are studied intensively. However, in silico tools for R-loop prediction are limited, and therefore, we have developed the R-loop tracker tool, which was implemented as a part of the DNA Analyser web server. This new tool is focused upon (1) prediction of R-loops in genomic DNA without length and sequence limitations; (2) integration of R-loop tracker results with other tools for nucleic acids analyses, including Genome Browser; (3) internal cross-evaluation of in silico results with experimental data, where available; (4) easy export and correlation analyses with other genome features and markers; and (5) enhanced visualization outputs. Our new R-loop tracker tool is freely accessible on the web pages of DNA Analyser tools, and its implementation on the web-based server allows effective analyses not only for DNA segments but also for full chromosomes and genomes.

Recently, the quest for the mythical fountain of youth has produced extensive research programs that aim to extend the healthy lifespan of humans. Despite advances in our understanding of the aging process, the surprisingly extended lifespan and cancer resistance of some animal species remain unexplained. The p53 protein plays a crucial role in tumor suppression, tissue homeostasis, and aging. Long-lived, cancer-free African elephants have 20 copies of the TP 53 gene, including 19 retrogenes (38 alleles), which are partially active, whereas humans possess only one copy of TP 53 and have an estimated cancer mortality rate of 11-25%. The mechanism through which p53 contributes to the resolution of the Peto's paradox in Animalia remains vague. Thus, in this work, we took advantage of the available datasets and inspected the p53 amino acid sequence of phylogenetically related organisms that show variations in their lifespans. We discovered new correlations between specific amino acid deviations in p53 and the lifespans across different animal species. We found that species with extended lifespans have certain characteristic amino acid substitutions in the p53 DNA-binding domain that alter its function, as depicted from the Phenotypic Annotation of p53 Mutations, using the PROVEAN tool or SWISS-MODEL workflow. In addition, the loop 2 region of the human p53 DNA-binding domain was identified as the longest region that was associated with longevity. The 3D model revealed variations in the loop 2 structure in long-lived species when compared with human p53. Our findings show a direct association between specific amino acid residues in p53 protein, changes in p53 functionality, and the extended animal lifespan, and further highlight the importance of p53 protein in aging.

DNA is fundamentally important for all cellular organisms due to its role as a store of hereditary genetic information. The precise and accurate regulation of gene transcription depends primarily on promoters, which vary significantly within and between genomes. Some promoters are rich in specific types of bases, while others have more varied, complex sequence characteristics. However, it is not only base sequence but also epigenetic modifications and altered DNA structure that regulate promoter activity. Significantly, many promoters across all organisms contain sequences that can form intrastrand hairpins (cruciforms) or four-stranded structures (G-quadruplex or i-motif). In this review we integrate recent studies on promoter regulation that highlight the importance of DNA structure in the evolutionary adaptation of promoter sequences., Competing Interests: Declaration of Interests The authors have no interests to declare., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

One of the changes brought about by Wallerian degeneration distal to nerve injury is disintegration of axonal mitochondria and consequent leakage of mitochondrial DNA (mtDNA)-the natural ligand for the toll-like receptor 9 (TLR9). RT-PCR and immunohistochemical or Western blot analyses were used to detect TLR9 mRNA and protein respectively in the lumbar (L4-L5) and cervical (C7-C8) dorsal root ganglia (DRG) ipsilateral and contralateral to a sterile unilateral sciatic nerve compression or transection. The unilateral sciatic nerve lesions led to bilateral increases in levels of both TLR9 mRNA and protein not only in the lumbar but also in the remote cervical DRG compared with naive or sham-operated controls. This upregulation of TLR9 was linked to activation of the Nuclear Factor kappa B (NFκB) and nuclear translocation of the Signal Transducer and Activator of Transcription 3 (STAT3), implying innate neuronal immune reaction and a pro-regenerative state in uninjured primary sensory neurons of the cervical DRG. The relationship of TLR9 to the induction of a pro-regenerative state in the cervical DRG neurons was confirmed by the shorter lengths of regenerated axons distal to ulnar nerve crush following a previous sciatic nerve lesion and intrathecal chloroquine injection compared with control rats. The results suggest that a systemic innate immune reaction not only triggers the regenerative state of axotomized DRG neurons but also induces a pro-regenerative state further along the neural axis after unilateral nerve injury.

G-quadruplexes contribute to the regulation of key molecular processes. Their utilization for antiviral therapy is an emerging field of contemporary research. Here we present comprehensive analyses of the presence and localization of putative G-quadruplex forming sequences (PQS) in all viral genomes currently available in the NCBI database (including subviral agents). The G4Hunter algorithm was applied to a pool of 11,000 accessible viral genomes representing 350 Mbp in total. PQS frequencies differ across evolutionary groups of viruses, and are enriched in repeats, replication origins, 5'UTRs and 3'UTRs. Importantly, PQS presence and localization is connected to viral lifecycles and corresponds to the type of viral infection rather than to nucleic acid type; while viruses routinely causing persistent infections in Metazoa hosts are enriched for PQS, viruses causing acute infections are significantly depleted for PQS. The unique localization of PQS identifies the importance of G-quadruplex-based regulation of viral replication and life cycle, providing a tool for potential therapeutic targeting., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Telomerase RNA (TR) carries the template for synthesis of telomere DNA and provides a scaffold for telomerase assembly. Fungal TRs are long and have been compared to higher eukaryotes, where they show considerable diversity within phylogenetically close groups. TRs of several Saccharomycetaceae were recently identified, however, many of these remained uncharacterised in the template region. Here we show that this is mainly due to high variability in telomere sequence. We predicted the telomere sequences using Tandem Repeats Finder and then we identified corresponding putative template regions in TR candidates. Remarkably long telomere units and the corresponding putative TRs were found in Tetrapisispora species. Notably, variable lengths of the annealing sequence of the template region (1-10 nt) were found. Consequently, species with the same telomere sequence may not harbour identical TR templates. Thus, TR sequence alone can be used to predict a template region and telomere sequence, but not to determine these exactly. A conserved feature of telomere sequences, tracts of adjacent Gs, led us to test the propensity of individual telomere sequences to form G4. The results show highly diverse values of G4-propensity, indicating the lack of ubiquitous conservation of this feature across Saccharomycetaceae.

Fungal infections cause >1 million deaths annually and the emergence of antifungal resistance has prompted the exploration for novel antifungal targets. Quadruplexes are four-stranded nucleic acid secondary structures, which can regulate processes such as transcription, translation, replication and recombination. They are also found in genes linked to virulence in microbes, and ligands that bind to quadruplexes can eliminate drug-resistant pathogens. Using a computational approach, we quantified putative quadruplex-forming sequences (PQS) in 1359 genomes across the fungal kingdom and explored their presence in genes related to virulence, drug resistance and biological processes associated with pathogenicity in Aspergillus fumigatus . Here we present the largest analysis of PQS in fungi and identify significant heterogeneity of these sequences throughout phyla, genera and species. PQS were genetically conserved in Aspergillus spp. and frequently pathogenic species appeared to contain fewer PQS than their lesser/non-pathogenic counterparts. GO-term analysis identified that PQS-containing genes were involved in processes linked with virulence such as zinc ion binding, the biosynthesis of secondary metabolites and regulation of transcription in A. fumigatus . Although the genome frequency of PQS was lower in A. fumigatus , PQS could be found enriched in genes involved in virulence, and genes upregulated during germination and hypoxia. Moreover, PQS were found in genes involved in drug resistance. Quadruplexes could have important roles within fungal biology and virulence, but their roles require further elucidation.

The importance of gene expression regulation in viruses based upon G-quadruplex may point to its potential utilization in therapeutic targeting. Here, we present analyses as to the occurrence of putative G-quadruplex-forming sequences (PQS) in all reference viral dsDNA genomes and evaluate their dependence on PQS occurrence in host organisms using the G4Hunter tool. PQS frequencies differ across host taxa without regard to GC content. The overlay of PQS with annotated regions reveals the localization of PQS in specific regions. While abundance in some, such as repeat regions, is shared by all groups, others are unique. There is abundance within introns of Eukaryota-infecting viruses, but depletion of PQS in introns of bacteria-infecting viruses. We reveal a significant positive correlation between PQS frequencies in dsDNA viruses and corresponding hosts from archaea, bacteria, and eukaryotes. A strong relationship between PQS in a virus and its host indicates their close coevolution and evolutionarily reciprocal mimicking of genome organization.

SARS-CoV-2 is an intensively investigated virus from the order Nidovirales (Coronaviridae family) that causes COVID-19 disease in humans. Through enormous scientific effort, thousands of viral strains have been sequenced to date, thereby creating a strong background for deep bioinformatics studies of the SARS-CoV-2 genome. In this study, we inspected high-frequency mutations of SARS-CoV-2 and carried out systematic analyses of their overlay with inverted repeat (IR) loci and CpG islands. The main conclusion of our study is that SARS-CoV-2 hot-spot mutations are significantly enriched within both IRs and CpG island loci. This points to their role in genomic instability and may predict further mutational drive of the SARS-CoV-2 genome. Moreover, CpG islands are strongly enriched upstream from viral ORFs and thus could play important roles in transcription and the viral life cycle. We hypothesize that hypermethylation of these loci will decrease the transcription of viral ORFs and could therefore limit the progression of the disease., (© The Author(s) 2020. Published by Oxford University Press.)

Background: Influenza viruses are dangerous pathogens. Seventy-Seven genomes of recently emerged genotype 4 reassortant Eurasian avian-like H1N1 virus (G4-EA-H1N1) are currently available. We investigated the presence and variation of potential G-quadruplex forming sequences (PQS), which can serve as targets for antiviral treatment., Results: PQS were identified in all 77 genomes. The total number of PQS in G4-EA-H1N1 genomes was 571. Interestingly, the number of PQS per genome in individual close relative viruses varied from 4 to 12. PQS were not randomly distributed in the 8 segments of the G4-EA-H1N1 genome, the highest frequency of PQS being found in the NP segment (1.39 per 1000 nt), which is considered a potential target for antiviral therapy. In contrast, no PQS was found in the NS segment. Analyses of variability pointed the importance of some PQS; even if genome variation of influenza virus is extreme, the PQS with the highest G4Hunter score is the most conserved in all tested genomes. G-quadruplex formation in vitro was experimentally confirmed using spectroscopic methods., Conclusions: The results presented here hint several G-quadruplex-forming sequences in G4-EA-H1N1 genomes, that could provide good therapeutic targets.

Subarachnoid hemorrhage is a specific, life-threatening form of hemorrhagic stroke linked to high morbidity and mortality. It has been found that the choroid plexus of the brain ventricles forming the blood-cerebrospinal fluid barrier plays an important role in subarachnoid hemorrhage pathophysiology. Heme oxygenase-1 and biliverdin reductase are two of the key enzymes of the hemoglobin degradation cascade. Therefore, the aim of present study was to investigate changes in protein levels of heme oxygenase-1 and biliverdin reductase in the rat choroid plexus after experimental subarachnoid hemorrhage induced by injection of non-heparinized autologous blood to the cisterna magna. Artificial cerebrospinal fluid of the same volume as autologous blood was injected to mimic increased intracranial pressure in control rats. Immunohistochemical and Western blot analyses were used to monitor changes in the of heme oxygenase-1 and biliverdin reductase levels in the rat choroid plexus after induction of subarachnoid hemorrhage or artificial cerebrospinal fluid application for 1, 3, and 7 days. We found increased levels of heme oxygenase-1 and biliverdin reductase protein in the choroid plexus over the entire period following subarachnoid hemorrhage induction. The level of heme oxygenase-1 was the highest early (1 and 3 days) after subarachnoid hemorrhage indicating its importance in hemoglobin degradation. Increased levels of heme oxygenase-1 were also observed in the choroid plexus epithelial cells at all time points after application of artificial cerebrospinal fluid. Biliverdin reductase protein was detected mainly in the choroid plexus epithelial cells, with levels gradually increasing during subarachnoid hemorrhage. Our results suggest that heme oxygenase-1 and biliverdin reductase are involved not only in hemoglobin degradation but probably also in protecting choroid plexus epithelial cells and the blood-cerebrospinal fluid barrier from the negative effects of subarachnoid hemorrhage., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Solár, Brázda, Levin, Zamani, Jančálek, Dubový and Joukal.)

The importance of unusual DNA structures in the regulation of basic cellular processes is an emerging field of research. Amongst local non-B DNA structures, G-quadruplexes (G4s) have gained in popularity during the last decade, and their presence and functional relevance at the DNA and RNA level has been demonstrated in a number of viral, bacterial, and eukaryotic genomes, including humans. Here, we performed the first systematic search of G4-forming sequences in all archaeal genomes available in the NCBI database. In this article, we investigate the presence and locations of G-quadruplex forming sequences using the G4Hunter algorithm. G-quadruplex-prone sequences were identified in all archaeal species, with highly significant differences in frequency, from 0.037 to 15.31 potential quadruplex sequences per kb. While G4 forming sequences were extremely abundant in Hadesarchaea archeon (strikingly, more than 50% of the Hadesarchaea archaeon isolate WYZ-LMO6 genome is a potential part of a G4-motif), they were very rare in the Parvarchaeota phylum. The presence of G-quadruplex forming sequences does not follow a random distribution with an over-representation in non-coding RNA, suggesting possible roles for ncRNA regulation. These data illustrate the unique and non-random localization of G-quadruplexes in Archaea.

Non-canonical nucleic acid structures play important roles in the regulation of molecular processes. Considering the importance of the ongoing coronavirus crisis, we decided to evaluate genomes of all coronaviruses sequenced to date (stated more broadly, the order Nidovirales ) to determine if they contain non-canonical nucleic acid structures. We discovered much evidence of putative G-quadruplex sites and even much more of inverted repeats (IRs) loci, which in fact are ubiquitous along the whole genomic sequence and indicate a possible mechanism for genomic RNA packaging. The most notable enrichment of IRs was found inside 5'UTR for IRs of size 12+ nucleotides, and the most notable enrichment of putative quadruplex sites (PQSs) was located before 3'UTR, inside 5'UTR, and before mRNA. This indicates crucial regulatory roles for both IRs and PQSs. Moreover, we found multiple G-quadruplex binding motifs in human proteins having potential for binding of SARS-CoV-2 RNA. Non-canonical nucleic acids structures in Nidovirales and in novel SARS-CoV-2 are therefore promising druggable structures that can be targeted and utilized in the future., (Copyright © 2020 Bartas, Brázda, Bohálová, Cantara, Volná, Stachurová, Malachová, Jagelská, Porubiaková, Červeň and Pečinka.)

Inverted repeats (IR) play important roles in specific DNA-dependent processes in simple prokaryotes to complex eukaryotes. They are recognized by a variety of proteins including restriction enzymes, helicases and transcription factors. We evaluate the presence and localization of IRs in all validated human promoter sequences within 1000 bp upstream and downstream of the transcription start site (TSS). The occurrence of 7 bp and longer IRs is located non-randomly in promoter regions, with enrichment within 200 bp upstream of the TSS. The highest frequency of IRs is just before TSS for repeats of 8 bp or longer. A comparison of promoters divided according to the occurrence of five individual promoter motifs shows unique location patterns of IRs. Principal component analyses and hierarchical clustering of IRs abundance demonstrated that they are depleted and/or not enriched in the promoters of stably expressed genes, but show significant enrichments for specific dynamically regulated biological pathways., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The importance of DNA structure in the regulation of basic cellular processes is an emerging field of research. Among local non-B DNA structures, inverted repeat (IR) sequences that form cruciforms and G-rich sequences that form G-quadruplexes (G4) are found in all prokaryotic and eukaryotic organisms and are targets for regulatory proteins. We analyzed IRs and G4 sequences in the genome of the most important biotechnology microorganism, S. cerevisiae. IR and G4-prone sequences are enriched in specific genomic locations and differ markedly between mitochondrial and nuclear DNA. While G4s are overrepresented in telomeres and regions surrounding tRNAs, IRs are most enriched in centromeres, rDNA, replication origins and surrounding tRNAs. Mitochondrial DNA is enriched in both IR and G4-prone sequences relative to the nuclear genome. This extensive analysis of local DNA structures adds to the emerging picture of their importance in genome maintenance, DNA replication and transcription of subsets of genes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

p53 is one of the most studied tumor suppressor proteins that plays an important role in basic biological processes including cell cycle, DNA damage response, apoptosis, and senescence. The human TP53 gene contains alternative promoters that produce N-terminally truncated proteins and can produce several isoforms due to alternative splicing. p53 function is realized by binding to a specific DNA response element (RE), resulting in the transactivation of target genes. Here, we evaluated the influence of quadruplex DNA structure on the transactivation potential of full-length and N-terminal truncated p53α isoforms in a panel of S. cerevisiae luciferase reporter strains. Our results show that a G-quadruplex prone sequence is not sufficient for transcription activation by p53α isoforms, but the presence of this feature in proximity to a p53 RE leads to a significant reduction of transcriptional activity and changes the dynamics between co-expressed p53α isoforms.

The p53 family of transcription factors plays key roles in development, genome stability, senescence and tumor development, and p53 is the most important tumor suppressor protein in humans. Although intensively investigated for many years, its initial evolutionary history is not yet fully elucidated. Using bioinformatic and structure prediction methods on current databases containing newly-sequenced genomes and transcriptomes, we present a detailed characterization of p53 family homologs in remote members of the Holozoa group, in the unicellular clades Filasterea, Ichthyosporea and Corallochytrea. Moreover, we show that these newly characterized homologous sequences contain domains that can form structures with high similarity to the human p53 family DNA-binding domain, and some also show similarities to the oligomerization and SAM domains. The presence of these remote homologs demonstrates an ancient origin of the p53 protein family.

The tumor suppressor functions of p53 and its roles in regulating the cell cycle, apoptosis, senescence, and metabolism are accomplished mainly by its interactions with DNA. p53 works as a transcription factor for a significant number of genes. Most p53 target genes contain so-called p53 response elements in their promoters, consisting of 20 bp long canonical consensus sequences. Compared to other transcription factors, which usually bind to one concrete and clearly defined DNA target, the p53 consensus sequence is not strict, but contains two repeats of a 5'RRRCWWGYYY3' sequence; therefore it varies remarkably among target genes. Moreover, p53 binds also to DNA fragments that at least partially and often completely lack this consensus sequence. p53 also binds with high affinity to a variety of non-B DNA structures including Holliday junctions, cruciform structures, quadruplex DNA, triplex DNA, DNA loops, bulged DNA, and hemicatenane DNA. In this review, we summarize information of the interactions of p53 with various DNA targets and discuss the functional consequences of the rich world of p53 DNA binding targets for its complex regulatory functions.