Mycobacterium tuberculosis O(6)-methylguanine-DNA methyltransferase (MtOGT) contributes to protect the bacterial GC-rich genome against the pro-mutagenic potential of O(6)-methylated guanine in DNA. Several strains of M. tuberculosis found worldwide encode a point-mutated O(6)-methylguanine-DNA methyltransferase (OGT) variant (MtOGT-R37L), which displays an arginine-to-leucine substitution at position 37 of the poorly functionally characterized N-terminal domain of the protein. Although the impact of this mutation on the MtOGT activity has not yet been proved in vivo, we previously demonstrated that a recombinant MtOGT-R37L variant performs a suboptimal alkylated-DNA repair in vitro, suggesting a direct role for the Arg(37)-bearing region in catalysis. The crystal structure of MtOGT complexed with modified DNA solved in the present study reveals details of the protein-protein and protein-DNA interactions occurring during alkylated-DNA binding, and the protein capability also to host unmodified bases inside the active site, in a fully extrahelical conformation. Our data provide the first experimental picture at the atomic level of a possible mode of assembling three adjacent MtOGT monomers on the same monoalkylated dsDNA molecule, and disclose the conformational flexibility of discrete regions of MtOGT, including the Arg(37)-bearing random coil. This peculiar structural plasticity of MtOGT could be instrumental to proper protein clustering at damaged DNA sites, as well as to protein-DNA complexes disassembling on repair.

• Klíčová slova
• DNA repair, DNA-binding protein, Mycobacterium tuberculosis, O6-methylguanine-DNA methyltransferase, co-operativity, crystal structure,
• MeSH
• bakteriální proteiny chemie   genetika   MeSH
• bodová mutace genetika   MeSH
• krystalografie MeSH
• Mycobacterium tuberculosis genetika   MeSH
• O(6)-methylguanin-DNA-methyltransferasa chemie   genetika   MeSH
• poškození DNA genetika   MeSH
• sekundární struktura proteinů MeSH
• terciární struktura proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• O(6)-methylguanin-DNA-methyltransferasa 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.

• Klíčová slova
• DNA, RNA, SELEX, aptamer, artificial evolution, deoxyribozyme, in vitro selection, nucleic acids, ribozyme, secondary structure, synthetic biology,
• 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
• Názvy látek
• aptamery nukleotidové MeSH
• DNA katalytická MeSH
• RNA katalytická MeSH

Interaction with the DNA minor groove is a significant contributor to specific sequence recognition in selected families of DNA-binding proteins. Based on a statistical analysis of 3D structures of protein-DNA complexes, we propose that distortion of the DNA minor groove resulting from interactions with hydrophobic amino acid residues is a universal element of protein-DNA recognition. We provide evidence to support this by associating each DNA minor groove-binding amino acid residue with the local dimensions of the DNA double helix using a novel algorithm. The widened DNA minor grooves are associated with high GC content. However, some AT-rich sequences contacted by hydrophobic amino acids (e.g., phenylalanine) display extreme values of minor groove width as well. For a number of hydrophobic amino acids, distinct secondary structure preferences could be identified for residues interacting with the widened DNA minor groove. These results hold even after discarding the most populous families of minor groove-binding proteins.

• Klíčová slova
• DNA shape, hydrophobic, indirect readout, minor groove, protein–DNA interaction, specific recognition,
• MeSH
• algoritmy MeSH
• aminokyselinové motivy MeSH
• aminokyseliny chemie   MeSH
• Arabidopsis metabolismus   MeSH
• DNA vazebné proteiny metabolismus   MeSH
• DNA chemie   MeSH
• fenylalanin chemie   MeSH
• hydrofobní a hydrofilní interakce * MeSH
• konformace nukleové kyseliny MeSH
• kyselina glutamová chemie   MeSH
• lidé MeSH
• proteiny chemie   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• sekundární struktura proteinů MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aminokyseliny MeSH
• DNA vazebné proteiny MeSH
• DNA MeSH
• fenylalanin MeSH
• kyselina glutamová MeSH
• proteiny MeSH

Secondary structure elements (SSEs) are inherent parts of protein structures, and their arrangement is characteristic for each protein family. Therefore, annotation of SSEs can facilitate orientation in the vast number of homologous structures which is now available for many protein families. It also provides a way to identify and annotate the key regions, like active sites and channels, and subsequently answer the key research questions, such as understanding of molecular function and its variability.This chapter introduces the concept of SSE annotation and describes the workflow for obtaining SSE annotation for the members of a selected protein family using program SecStrAnnotator.

• Klíčová slova
• Annotation, Protein domain, Protein family, SecStrAnnotator, Secondary structure, Secondary structure assignment, Secondary structure elements, Structural alignment,
• MeSH
• algoritmy MeSH
• aminokyselinové motivy * MeSH
• anotace sekvence metody   MeSH
• katalytická doména genetika   MeSH
• proteiny chemie   genetika   MeSH
• software MeSH
• výpočetní biologie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny MeSH

SUMMARY: We present the cpPredictor webserver that implements a novel template-based method for prediction of secondary structure of RNA. The method outperforms available prediction methods as it uses RNA structures of related molecules, either predicted or experimentally identified, as structural templates. The server aims at three major tasks: i) prediction of RNA secondary structures that are difficult to predict by available methods, ii) characterization of uncharacterized RNAs as compatible or incompatible with a chosen template structure and iii) an identification of the most relevant structure among different candidate structures of a single RNA ambiguously predicted by available methods. The web server is accompanied with a comprehensive documentation. AVAILABILITY AND IMPLEMENTATION: The web server is freely available at http://cppredictor.elixir-czech.cz/. The source code of the cpPredictor algorithm is freely available from the webserver under the Apache License, Version 2.0.

• MeSH
• algoritmy MeSH
• internet MeSH
• konformace nukleové kyseliny * MeSH
• RNA MeSH
• sekundární struktura proteinů MeSH
• sekvenční analýza RNA MeSH
• software * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• RNA MeSH

BACKGROUND: Visualization of RNA secondary structures is a complex task, and, especially in the case of large RNA structures where the expected layout is largely habitual, the existing visualization tools often fail to produce suitable visualizations. This led us to the idea to use existing layouts as templates for the visualization of new RNAs similarly to how templates are used in homology-based structure prediction. RESULTS: This article introduces Traveler, a software tool enabling visualization of a target RNA secondary structure using an existing layout of a sufficiently similar RNA structure as a template. Traveler is based on an algorithm which converts the target and template structures into corresponding tree representations and utilizes tree edit distance coupled with layout modification operations to transform the template layout into the target one. Traveler thus accepts a pair of secondary structures and a template layout and outputs a layout for the target structure. CONCLUSIONS: Traveler is a command-line open source tool able to quickly generate layouts for even the largest RNA structures in the presence of a sufficiently similar layout. It is available at http://github.com/davidhoksza/traveler .

• Klíčová slova
• RNA secondary structure, Software tool, Template-based modeling, Visualization,
• MeSH
• algoritmy MeSH
• konformace nukleové kyseliny MeSH
• RNA chemie   MeSH
• software * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• RNA MeSH

To maintain genome integrity, segmented double-stranded RNA viruses of the Reoviridae family must accurately select and package a complete set of up to a dozen distinct genomic RNAs. It is thought that the high fidelity segmented genome assembly involves multiple sequence-specific RNA-RNA interactions between single-stranded RNA segment precursors. These are mediated by virus-encoded non-structural proteins with RNA chaperone-like activities, such as rotavirus (RV) NSP2 and avian reovirus σNS. Here, we compared the abilities of NSP2 and σNS to mediate sequence-specific interactions between RV genomic segment precursors. Despite their similar activities, NSP2 successfully promotes inter-segment association, while σNS fails to do so. To understand the mechanisms underlying such selectivity in promoting inter-molecular duplex formation, we compared RNA-binding and helix-unwinding activities of both proteins. We demonstrate that octameric NSP2 binds structured RNAs with high affinity, resulting in efficient intramolecular RNA helix disruption. Hexameric σNS oligomerizes into an octamer that binds two RNAs, yet it exhibits only limited RNA-unwinding activity compared to NSP2. Thus, the formation of intersegment RNA-RNA interactions is governed by both helix-unwinding capacity of the chaperones and stability of RNA structure. We propose that this protein-mediated RNA selection mechanism may underpin the high fidelity assembly of multi-segmented RNA genomes in Reoviridae.

• MeSH
• genom virový genetika   MeSH
• konformace nukleové kyseliny MeSH
• molekulární chaperony chemie   genetika   metabolismus   MeSH
• molekulární modely MeSH
• proteiny vázající RNA chemie   genetika   metabolismus   MeSH
• ptačí orthoreovirus genetika   metabolismus   MeSH
• RNA virová chemie   genetika   metabolismus   MeSH
• sekundární struktura proteinů MeSH
• sekvence nukleotidů MeSH
• vazba proteinů MeSH
• virové nestrukturální proteiny chemie   genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• molekulární chaperony MeSH
• NS35 protein, rotavirus MeSH Prohlížeč
• proteiny vázající RNA MeSH
• RNA virová MeSH
• virové nestrukturální proteiny MeSH

Naphthoquinones represent the group of plant secondary metabolites with cytotoxic properties based on their ability to generate reactive oxygen species and interfere with the processes of cell respiration. Due to this fact, the possible cytotoxic mechanisms on cellular and subcellular levels are investigated intensively. There are many targets of cytotoxic action on the cellular level; however, DNA is a critical target of many cytotoxic compounds. Due to the cytotoxic properties of naphthoquinones, it is necessary to study the processes of naphthoquinones, DNA interactions (1,4-naphthoquinone, binapthoquinone, juglone, lawsone, plumbagin), especially by using modern analytical techniques. In our work, the Raman spectroscopy was used to determine the possible binding sites of the naphthoquinones on the DNA and to characterize the bond of naphthoquinone to DNA. Experimental data reveals the relationships between the perturbations of structure-sensitive Raman bands and the types of the naphthoquinones involved. The modification of DNA by the studied naphthoquinones leads to the nonspecific interaction, which causes the transition of B-DNA into A-DNA conformation. The change of the B-conformation of DNA for all measured DNA modified by naphthoquinones except plumbagin is obvious.

• MeSH
• B-DNA chemie   metabolismus   MeSH
• DNA chemie   metabolismus   MeSH
• naftochinony chemie   metabolismus   MeSH
• Ramanova spektroskopie MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• rostliny chemie   metabolismus   MeSH
• sekundární metabolismus * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• B-DNA MeSH
• DNA MeSH
• juglone MeSH Prohlížeč
• lawsone MeSH Prohlížeč
• naftochinony MeSH
• plumbagin MeSH Prohlížeč
• reaktivní formy kyslíku MeSH

Various base lesions continuously form in cellular nucleic acids and the unrepaired lesions are promutagenic and procarcinogenic. Though natural base lesions have been extensively studied in double-stranded DNA models, these studies are only less than a decade old for non-canonical DNA models, such as quadruplexes. Here we present a report on the effects of three frequently occurring natural lesions that can form in the TTA loops on the structure of the human telomere quadruplex d[AG3(TTAG3)3]. We compared the effect of the abasic site and 8-oxoadenine replacing adenine and 5-hydroxymethyluracil substituting for thymine. The results showed that the three lesions impacted the stability and quadruplex folding in markedly different ways. The effects depended on the type of lesion and the position in the sequence. Analogous lesions of guanine in the G-tetrads extensively destabilized the quadruplex and the effects depended more on the position than on the type of lesion. The distinct effects of the loop substitutions as well as comparison of the modifications of the loops and the quadruplex tetrads are discussed in this communication.

• Klíčová slova
• Base modification, Circular dichroism spectroscopy, DNA secondary structure, Guanine quadruplex, Oxidative damage,
• MeSH
• cirkulární dichroismus MeSH
• G-kvadruplexy * MeSH
• konformace nukleové kyseliny * MeSH
• lidé MeSH
• molekulární modely * MeSH
• poškození DNA genetika   MeSH
• telomery chemie   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Thymidine triphosphate bearing benzylidene-tetrahydroxanthylium near-IR fluorophore linked to the 5-methyl group via triazole was synthesized through the CuAAC reaction and was used for polymerase synthesis of labelled DNA probes. The fluorophore lights up upon incorporation to DNA (up to 348-times) presumably due to interactions in major groove and the fluorescence further increases in the single-stranded oligonucleotide. The labelled dsDNA senses binding of small molecules and proteins by a strong decrease of fluorescence. The nucleotide was used as a light-up building block in real-time PCR for detection of SARS-CoV-2 virus.

• Klíčová slova
• DNA, fluorescence, nucleotides, real-time PCR,
• MeSH
• COVID-19 * MeSH
• DNA sondy MeSH
• lidé MeSH
• nukleotidy MeSH
• replikace DNA * MeSH
• SARS-CoV-2 MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA sondy MeSH
• nukleotidy MeSH