Extensive Bioinformatics Analyses Reveal a Phylogenetically Conserved Winged Helix (WH) Domain (Zτ) of Topoisomerase IIα, Elucidating Its Very High Affinity for Left-Handed Z-DNA and Suggesting Novel Putative Functions.

The dynamic processes operating on genomic DNA, such as gene expression and cellular division, lead inexorably to topological challenges in the form of entanglements, catenanes, knots, "bubbles", R-loops, and other outcomes of supercoiling and helical disruption. The resolution of toxic topological stress is the function attributed to DNA topoisomerases. A prominent example is the negative supercoiling (nsc) trailing processive enzymes such as DNA and RNA polymerases. The multiple equilibrium states that nscDNA can adopt by redistribution of helical twist and writhe include the left-handed double-helical conformation known as Z-DNA. Thirty years ago, one of our labs isolated a protein from Drosophila cells and embryos with a 100-fold greater affinity for Z-DNA than for B-DNA, and identified it as topoisomerase II (gene Top2, orthologous to the human UniProt proteins TOP2A and TOP2B). GTP increased the affinity and selectivity for Z-DNA even further and also led to inhibition of the isomerase enzymatic activity. An allosteric mechanism was proposed, in which topoII acts as a Z-DNA-binding protein (ZBP) to stabilize given states of topological (sub)domains and associated multiprotein complexes. We have now explored this possibility by comprehensive bioinformatic analyses of the available protein sequences of topoII representing organisms covering the whole tree of life. Multiple alignment of these sequences revealed an extremely high level of evolutionary conservation, including a winged-helix protein segment, here denoted as Zτ, constituting the putative structural homolog of Zα, the canonical Z-DNA/Z-RNA binding domain previously identified in the interferon-inducible RNA Adenosine-to-Inosine-editing deaminase, ADAR1p150. In contrast to Zα, which is separate from the protein segment responsible for catalysis, Zτ encompasses the active site tyrosine of topoII; a GTP-binding site and a GxxG sequence motif are in close proximity. Quantitative Zτ-Zα similarity comparisons and molecular docking with interaction scoring further supported the "B-Z-topoII hypothesis" and has led to an expanded mechanism for topoII function incorporating the recognition of Z-DNA segments ("Z-flipons") as an inherent and essential element. We further propose that the two Zτ domains of the topoII homodimer exhibit a single-turnover "conformase" activity on given G(ate) B-DNA segments ("Z-flipins"), inducing their transition to the left-handed Z-conformation. Inasmuch as the topoII-Z-DNA complexes are isomerase inactive, we infer that they fulfill important structural roles in key processes such as mitosis. Topoisomerases are preeminent targets of anti-cancer drug discovery, and we anticipate that detailed elucidation of their structural-functional interactions with Z-DNA and GTP will facilitate the design of novel, more potent and selective anti-cancer chemotherapeutic agents.

Identification of annotation artifacts concerning the chalcone synthase (CHS).

OBJECTIVE: Chalcone synthase (CHS) catalyzes the initial step of the flavonoid biosynthesis. The CHS encoding gene is well studied in numerous plant species. Rapidly growing sequence databases contain hundreds of CHS entries that are the result of automatic annotation. In this study, we evaluated apparent multiplication of CHS domains in CHS gene models of four plant species. MAIN FINDINGS: CHS genes with an apparent triplication of the CHS domain encoding part were discovered through database searches. Such genes were found in Macadamia integrifolia, Musa balbisiana, Musa troglodytarum, and Nymphaea colorata. A manual inspection of the CHS gene models in these four species with massive RNA-seq data suggests that these gene models are the result of artificial fusions in the annotation process. While there are hundreds of seemingly correct CHS records in the databases, it is not clear why these annotation artifacts appeared.

Variability of Inverted Repeats in All Available Genomes of Bacteria.

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.

Beyond the Primary Structure of Nucleic Acids: Potential Roles of Epigenetics and Noncanonical Structures in the Regulations of Plant Growth and Stress Responses.

Epigenetics deals with changes in gene expression that are not caused by modifications in the primary sequence of nucleic acids. These changes beyond primary structures of nucleic acids not only include DNA/RNA methylation, but also other reversible conversions, together with histone modifications or RNA interference. In addition, under particular conditions (such as specific ion concentrations or protein-induced stabilization), the right-handed double-stranded DNA helix (B-DNA) can form noncanonical structures commonly described as "non-B DNA" structures. These structures comprise, for example, cruciforms, i-motifs, triplexes, and G-quadruplexes. Their formation often leads to significant differences in replication and transcription rates. Noncanonical RNA structures have also been documented to play important roles in translation regulation and the biology of noncoding RNAs. In human and animal studies, the frequency and dynamics of noncanonical DNA and RNA structures are intensively investigated, especially in the field of cancer research and neurodegenerative diseases. In contrast, noncanonical DNA and RNA structures in plants have been on the fringes of interest for a long time and only a few studies deal with their formation, regulation, and physiological importance for plant stress responses. Herein, we present a review focused on the main fields of epigenetics in plants and their possible roles in stress responses and signaling, with special attention dedicated to noncanonical DNA and RNA structures.

New Low Morphine Opium Poppy Genotype Obtained by TILLING Approach.

The opium poppy's ability to produce various alkaloids is both useful and problematic. Breeding of new varieties with varying alkaloid content is therefore an important task. In this paper, the breeding technology of new low morphine poppy genotypes, based on a combination of a TILLING approach and single-molecule real-time NGS sequencing, is presented. Verification of the mutants in the TILLING population was obtained using RT-PCR and HPLC methods. Only three of the single-copy genes of the morphine pathway among the eleven genes were used for the identification of mutant genotypes. Point mutations were obtained only in one gene (CNMT) while an insertion was obtained in the other (SalAT). Only a few expected transition SNPs from G:C to A:T were obtained. In the low morphine mutant genotype, the production of morphine was decreased to 0.1% from 1.4% in the original variety. A comprehensive description of the breeding process, a basic characterization of the main alkaloid content, and a gene expression profile for the main alkaloid-producing genes is provided. Difficulties with the TILLING approach are also described and discussed.

Analysis of G-Quadruplex-Forming Sequences in Drought Stress-Responsive Genes, and Synthesis Genes of Phenolic Compounds in Arabidopsis thaliana.

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.

What Do We Know about Barley miRNAs?

Plant miRNAs are powerful regulators of gene expression at the post-transcriptional level, which was repeatedly proved in several model plant species. miRNAs are considered to be key regulators of many developmental, homeostatic, and immune processes in plants. However, our understanding of plant miRNAs is still limited, despite the fact that an increasing number of studies have appeared. This systematic review aims to summarize our current knowledge about miRNAs in spring barley (Hordeum vulgare), which is an important agronomical crop worldwide and serves as a common monocot model for studying abiotic stress responses as well. This can help us to understand the connection between plant miRNAs and (not only) abiotic stresses in general. In the end, some future perspectives and open questions are summarized.

The Newly Sequenced Genome of Pisum sativum Is Replete with Potential G-Quadruplex-Forming Sequences-Implications for Evolution and Biological Regulation.

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.

Regulation of Phenolic Compound Production by Light Varying in Spectral Quality and Total Irradiance.

Photosynthetically active radiation (PAR) is an important environmental cue inducing the production of many secondary metabolites involved in plant oxidative stress avoidance and tolerance. To examine the complex role of PAR irradiance and specific spectral components on the accumulation of phenolic compounds (PheCs), we acclimated spring barley (Hordeum vulgare) to different spectral qualities (white, blue, green, red) at three irradiances (100, 200, 400 µmol m-2 s-1). We confirmed that blue light irradiance is essential for the accumulation of PheCs in secondary barley leaves (in UV-lacking conditions), which underpins the importance of photoreceptor signals (especially cryptochrome). Increasing blue light irradiance most effectively induced the accumulation of B-dihydroxylated flavonoids, probably due to the significantly enhanced expression of the F3'H gene. These changes in PheC metabolism led to a steeper increase in antioxidant activity than epidermal UV-A shielding in leaf extracts containing PheCs. In addition, we examined the possible role of miRNAs in the complex regulation of gene expression related to PheC biosynthesis.

Unheeded SARS-CoV-2 proteins? A deep look into negative-sense RNA.

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.

Are There Hidden Genes in DNA/RNA Vaccines?

Due to the fast global spreading of the Severe Acute Respiratory Syndrome Coronavirus - 2 (SARS-CoV-2), prevention and treatment options are direly needed in order to control infection-related morbidity, mortality, and economic losses. Although drug and inactivated and attenuated virus vaccine development can require significant amounts of time and resources, DNA and RNA vaccines offer a quick, simple, and cheap treatment alternative, even when produced on a large scale. The spike protein, which has been shown as the most antigenic SARS-CoV-2 protein, has been widely selected as the target of choice for DNA/RNA vaccines. Vaccination campaigns have reported high vaccination rates and protection, but numerous unintended effects, ranging from muscle pain to death, have led to concerns about the safety of RNA/DNA vaccines. In parallel to these studies, several open reading frames (ORFs) have been found to be overlapping SARS-CoV-2 accessory genes, two of which, ORF2b and ORF-Sh, overlap the spike protein sequence. Thus, the presence of these, and potentially other ORFs on SARS-CoV-2 DNA/RNA vaccines, could lead to the translation of undesired proteins during vaccination. Herein, we discuss the translation of overlapping genes in connection with DNA/RNA vaccines. Two mRNA vaccine spike protein sequences, which have been made publicly-available, were compared to the wild-type sequence in order to uncover possible differences in putative overlapping ORFs. Notably, the Moderna mRNA-1273 vaccine sequence is predicted to contain no frameshifted ORFs on the positive sense strand, which highlights the utility of codon optimization in DNA/RNA vaccine design to remove undesired overlapping ORFs. Since little information is available on ORF2b or ORF-Sh, we use structural bioinformatics techniques to investigate the structure-function relationship of these proteins. The presence of putative ORFs on DNA/RNA vaccine candidates implies that overlapping genes may contribute to the translation of smaller peptides, potentially leading to unintended clinical outcomes, and that the protein-coding potential of DNA/RNA vaccines should be rigorously examined prior to administration.

Searching for New Z-DNA/Z-RNA Binding Proteins Based on Structural Similarity to Experimentally Validated Zα Domain.

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.

Transcriptomic and Proteomic Analysis of Drought Stress Response in Opium Poppy Plants during the First Week of Germination.

Water deficiency is one of the most significant abiotic stresses that negatively affects growth and reduces crop yields worldwide. Most research is focused on model plants and/or crops which are most agriculturally important. In this research, drought stress was applied to two drought stress contrasting varieties of Papaver somniferum (the opium poppy), a non-model plant species, during the first week of its germination, which differ in responses to drought stress. After sowing, the poppy seedlings were immediately subjected to drought stress for 7 days. We conducted a large-scale transcriptomic and proteomic analysis for drought stress response. At first, we found that the transcriptomic and proteomic profiles significantly differ. However, the most significant findings are the identification of key genes and proteins with significantly different expressions relating to drought stress, e.g., the heat-shock protein family, dehydration responsive element-binding transcription factors, ubiquitin E3 ligase, and others. In addition, metabolic pathway analysis showed that these genes and proteins were part of several biosynthetic pathways most significantly related to photosynthetic processes, and oxidative stress responses. A future study will focus on a detailed analysis of key genes and the development of selection markers for the determination of drought-resistant varieties and the breeding of new resistant lineages.

The Changes in the p53 Protein across the Animal Kingdom Point to Its Involvement in Longevity.

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 TP53 gene, including 19 retrogenes (38 alleles), which are partially active, whereas humans possess only one copy of TP53 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.

G-Quadruplex in Gene Encoding Large Subunit of Plant RNA Polymerase II: A Billion-Year-Old Story.

G-quadruplexes have long been perceived as rare and physiologically unimportant nucleic acid structures. However, several studies have revealed their importance in molecular processes, suggesting their possible role in replication and gene expression regulation. Pathways involving G-quadruplexes are intensively studied, especially in the context of human diseases, while their involvement in gene expression regulation in plants remains largely unexplored. Here, we conducted a bioinformatic study and performed a complex circular dichroism measurement to identify a stable G-quadruplex in the gene RPB1, coding for the RNA polymerase II large subunit. We found that this G-quadruplex-forming locus is highly evolutionarily conserved amongst plants sensu lato (Archaeplastida) that share a common ancestor more than one billion years old. Finally, we discussed a new hypothesis regarding G-quadruplexes interacting with UV light in plants to potentially form an additional layer of the regulatory network.

Evolution of Diverse Strategies for Promoter Regulation.

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.

Beta-lactam resistance development during the treatment processes of municipal wastewater treatment plants.

This work monitored the effect of a municipal and a village wastewater treatment plant (WWTP) technology on the fate of beta-lactam resistance genes in bacterial populations in different phases of the wastewater treatment process. In case of the municipal WWTP1, the bacteria possessing a high ampicillin resistance (minimal inhibitory concentration (MIC) values of 20 mg/mL) accumulated in the sedimentation tank, which was accompanied with a higher concentration of ampicillin in the wastewater samples (28.09 ng/L) and an increase in the relative abundance of the blaTEM gene in the bacterial population. However, an opposite trend was revealed with the blaNDM-1 gene, making the sedimentation processes of WWTP1 crucial only for the accumulation of the blaTEM gene. Similarly, the comparison with the WWTP2 showed that the accumulation of the ampicillin resistance in bacterial population probably depended on the WWTP technology and wastewater composition. Out of the four tested resistance genes (blaTEM, blaKPC, blaNDM-1, and blaOXA-48), blaTEM and blaNDM-1 genes were the only two detected in this study. According to NGS analysis of bacterial 16 S rRNA gene, Gammaproteobacteria dominated the ampicillin-resistant bacteria of the WWTP sedimentation tanks. Their relative abundance in the bacterial population also increased during the sedimentation processes in WWTP1. It could indicate the role of the bacterial taxon in ampicillin resistance accumulation in this WWTP and show that only 9.29% of the original bacterial population from the nitrification tank is involved in the documented shifts in beta-lactam resistance of the bacterial population.

Tracing dsDNA Virus-Host Coevolution through Correlation of Their G-Quadruplex-Forming Sequences.

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.

Analyses of viral genomes for G-quadruplex forming sequences reveal their correlation with the type of infection.

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.

Letter to the Editor: Significant mutation enrichment in inverted repeat sites of new SARS-CoV-2 strains.

In a recently published paper, we have found that SARS-CoV-2 hot-spot mutations are significantly associated with inverted repeat loci and CG dinucleotides. However, fast-spreading strains with new mutations (so-called mink farm mutations, England mutations and Japan mutations) have been recently described. We used the new datasets to check the positioning of mutation sites in genomes of the new SARS-CoV-2 strains. Using an open-access Palindrome analyzer tool, we found mutations in these new strains to be significantly enriched in inverted repeat loci.