Inverted repeat
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Non-canonical (non-B) DNA structures-e.g. bent DNA, hairpins, G-quadruplexes (G4s), Z-DNA, etc.-which form at certain sequence motifs (e.g. A-phased repeats, inverted repeats, etc.), have emerged as important regulators of cellular processes and drivers of genome evolution. Yet, they have been understudied due to their repetitive nature and potentially inaccurate sequences generated with short-read technologies. Here we comprehensively characterize such motifs in the long-read telomere-to-telomere (T2T) genomes of human, bonobo, chimpanzee, gorilla, Bornean orangutan, Sumatran orangutan, and siamang. Non-B DNA motifs are enriched at the genomic regions added to T2T assemblies and occupy 9%-15%, 9%-11%, and 12%-38% of autosomes and chromosomes X and Y, respectively. G4s and Z-DNA are enriched at promoters and enhancers, as well as at origins of replication. Repetitive sequences harbor more non-B DNA motifs than non-repetitive sequences, especially in the short arms of acrocentric chromosomes. Most centromeres and/or their flanking regions are enriched in at least one non-B DNA motif type, consistent with a potential role of non-B structures in determining centromeres. Our results highlight the uneven distribution of predicted non-B DNA structures across ape genomes and suggest their novel functions in previously inaccessible genomic regions.
- MeSH
- DNA * chemie genetika MeSH
- G-kvadruplexy MeSH
- genom lidský MeSH
- genom * MeSH
- Hominidae * genetika MeSH
- lidé MeSH
- nukleotidové motivy MeSH
- Pan troglodytes genetika MeSH
- repetitivní sekvence nukleových kyselin MeSH
- telomery * genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
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.
- MeSH
- COVID-19 * MeSH
- lidé MeSH
- mutace MeSH
- SARS-CoV-2 MeSH
- virus opičích neštovic * genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
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.
- MeSH
- Bacteria genetika MeSH
- fylogeneze MeSH
- genomika * MeSH
- lidé MeSH
- replikace DNA * MeSH
- sekvence nukleotidů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The secondary structure of nucleic acids containing quartets of guanines, termed G-quadruplexes, is known to regulate the transcription of many genes. Several G-quadruplexes can be formed in the HIV-1 long terminal repeat promoter region and their stabilization results in the inhibition of HIV-1 replication. Here, we identified helquat-based compounds as a new class of anti-HIV-1 inhibitors that inhibit HIV-1 replication at the stage of reverse transcription and provirus expression. Using Taq polymerase stop and FRET melting assays, we have demonstrated their ability to stabilize G-quadruplexes in the HIV-1 long-terminal repeat sequence. Moreover, these compounds were not binding to the general G-rich region, but rather to G-quadruplex-forming regions. Finally, docking and molecular dynamics calculations indicate that the structure of the helquat core greatly affects the binding mode to the individual G-quadruplexes. Our findings can provide useful information for the further rational design of inhibitors targeting G-quadruplexes in HIV-1.
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.
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.
- MeSH
- G-kvadruplexy * MeSH
- genom lidský MeSH
- inverze sekvence * MeSH
- lidé MeSH
- lidské chromozomy, pár 8 * MeSH
- sekvenční analýza DNA MeSH
- telomery * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
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.
DNA transposons are defined as repeated DNA sequences that can move within the host genome through the action of transposases. The transposon superfamily Merlin was originally found mainly in animal genomes. Here, we describe a global distribution of the Merlin in animals, fungi, plants and protists, reporting for the first time their presence in Rhodophyceae, Metamonada, Discoba and Alveolata. We identified a great variety of potentially active Merlin families, some containing highly imperfect terminal inverted repeats and internal tandem repeats. Merlin-related sequences with no evidence of mobilization capacity were also observed and may be products of domestication. The evolutionary trees support that Merlin is likely an ancient superfamily, with early events of diversification and secondary losses, although repeated re-invasions probably occurred in some groups, which would explain its diversity and discontinuous distribution. We cannot rule out the possibility that the Merlin superfamily is the product of multiple horizontal transfers of related prokaryotic insertion sequences. Moreover, this is the first account of a DNA transposon in kinetoplastid flagellates, with conserved Merlin transposase identified in Bodo saltans and Perkinsela sp., whereas it is absent in trypanosomatids. Based on the level of conservation of the transposase and overlaps of putative open reading frames with Merlin, we propose that in protists it may serve as a raw material for gene emergence.
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.
- MeSH
- COVID-19 virologie MeSH
- CpG ostrůvky * MeSH
- genom virový MeSH
- lidé MeSH
- metylace DNA MeSH
- mutace * MeSH
- SARS-CoV-2 genetika MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Methods of artificial evolution such as SELEX and in vitro selection have made it possible to isolate RNA and DNA motifs with a wide range of functions from large random sequence libraries. Once the primary sequence of a functional motif is known, the sequence space around it can be comprehensively explored using a combination of random mutagenesis and selection. However, methods to explore the sequence space of a secondary structure are not as well characterized. Here we address this question by describing a method to construct libraries in a single synthesis which are enriched for sequences with the potential to form a specific secondary structure, such as that of an aptamer, ribozyme, or deoxyribozyme. Although interactions such as base pairs cannot be encoded in a library using conventional DNA synthesizers, it is possible to modulate the probability that two positions will have the potential to pair by biasing the nucleotide composition at these positions. Here we show how to maximize this probability for each of the possible ways to encode a pair (in this study defined as A-U or U-A or C-G or G-C or G.U or U.G). We then use these optimized coding schemes to calculate the number of different variants of model stems and secondary structures expected to occur in a library for a series of structures in which the number of pairs and the extent of conservation of unpaired positions is systematically varied. Our calculations reveal a tradeoff between maximizing the probability of forming a pair and maximizing the number of possible variants of a desired secondary structure that can occur in the library. They also indicate that the optimal coding strategy for a library depends on the complexity of the motif being characterized. Because this approach provides a simple way to generate libraries enriched for sequences with the potential to form a specific secondary structure, we anticipate that it should be useful for the optimization and structural characterization of functional nucleic acid motifs.
- MeSH
- aptamery nukleotidové genetika MeSH
- DNA katalytická genetika MeSH
- genová knihovna * MeSH
- konformace nukleové kyseliny MeSH
- mutageneze MeSH
- nukleotidové motivy genetika MeSH
- obrácené repetice genetika MeSH
- párování bází MeSH
- pravděpodobnost MeSH
- řízená evoluce molekul metody MeSH
- RNA katalytická genetika MeSH
- syntetická biologie metody MeSH
- techniky in vitro MeSH
- Publikační typ
- časopisecké články MeSH
