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Genes To Cells : Devoted To Molecular &... Aug 2021Ribonucleotides incorporated in the genome are a source of endogenous DNA damage and also serve as signals for repair. Although recent advances of ribonucleotide...
Ribonucleotides incorporated in the genome are a source of endogenous DNA damage and also serve as signals for repair. Although recent advances of ribonucleotide detection by sequencing, the balance between incorporation and repair of ribonucleotides has not been elucidated. Here, we describe a competitive sequencing method, Ribonucleotide Scanning Quantification sequencing (RiSQ-seq), which enables absolute quantification of misincorporated ribonucleotides throughout the genome by background normalization and standard adjustment within a single sample. RiSQ-seq analysis of cells harboring wild-type DNA polymerases revealed that ribonucleotides were incorporated nonuniformly in the genome with a 3'-shifted distribution and preference for GC sequences. Although ribonucleotide profiles in wild-type and repair-deficient mutant strains showed a similar pattern, direct comparison of distinct ribonucleotide levels in the strains by RiSQ-seq enabled evaluation of ribonucleotide excision repair activity at base resolution and revealed the strand bias of repair. The distinct preferences of ribonucleotide incorporation and repair create vulnerable regions associated with indel hotspots, suggesting that repair at sites of ribonucleotide misincorporation serves to maintain genome integrity and that RiSQ-seq can provide an estimate of indel risk.
Topics: DNA; DNA Repair; Genome, Fungal; Mutation Rate; Ribonucleotides; Saccharomyces cerevisiae
PubMed: 33993586
DOI: 10.1111/gtc.12871 -
Nucleic Acids Research Jan 1977Viroids are uncoated infectious RNA molecules (MW 107 000-127 000) known as pathogens of certain higher plants. Thermodynamic and kinetic studies were carried out on...
Viroids are uncoated infectious RNA molecules (MW 107 000-127 000) known as pathogens of certain higher plants. Thermodynamic and kinetic studies were carried out on highly purified viroid preparations by applying UV-absorption melting analysis and temperature jump methods. The thermal denaturation of viroids is characterized by high thermal stability, high cooperativity and a high degree of base pairing. Two relaxation processes could be resolved; a process in the sec range could be evaluated as an independent all-or-none-transition with the following properties: reaction enthalpy= 550 kcal/mol, activation enthalpy of the dissociation = 470 kcal/mol; G : C content = 72 %. These data indicate the existence of an uninterrupted double helix of 52 base pairs. A process in the msec range involves 15 - 25 base pairs which are most probably distributed over several short double helical stretches. A tentative model for the secondary structure of viroids isproposed and the possible functional implications of their physicochemical properties are discussed.
Topics: Chemical Phenomena; Chemistry; Cytosine Nucleotides; Guanine Nucleotides; Kinetics; Nucleic Acid Conformation; Nucleic Acid Denaturation; Plant Diseases; Plants; RNA; Ribonucleotides; Temperature; Thermodynamics; Urea
PubMed: 866174
DOI: 10.1093/nar/4.1.177 -
The European Respiratory Journal Dec 2021We investigated the mechanisms by which N1-(β-d-ribofuranosyl)-5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an activator of AMP-activated protein kinase...
AIM
We investigated the mechanisms by which N1-(β-d-ribofuranosyl)-5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an activator of AMP-activated protein kinase (AMPK), decreases lung injury and mortality when administered to mice post exposure to bromine gas (Br).
METHODS
We exposed male C57BL/6 mice and heme oxygenase-1 (HO-1)-deficient (HO-1) and corresponding wild-type (WT) littermate mice to Br (600 ppm for 45 or 30 min, respectively) in environmental chambers and returned them to room air. AICAR was administered 6 h post exposure (10 mg·kg, intraperitoneal). We assessed survival, indices of lung injury, high mobility group box 1 (HMGB1) in the plasma, HO-1 levels in lung tissues and phosphorylation of AMPK and its upstream liver kinase B1 (LKB1). Rat alveolar type II epithelial (L2) cells and human club-like epithelial (H441) cells were also exposed to Br (100 ppm for 10 min). After 24 h we measured apoptosis and necrosis, AMPK and LKB1 phosphorylation, and HO-1 expression.
RESULTS
There was a marked downregulation of phosphorylated AMPK and LKB1 in lung tissues and in L2 and H441 cells post exposure. AICAR increased survival in C57BL/6 but not in HO-1 mice. In WT mice, AICAR decreased lung injury and restored phosphorylated AMPK and phosphorylated LKB1 to control levels and increased HO-1 levels in both lung tissues and cells exposed to Br. Treatment of L2 and H441 cells with small interfering RNAs against nuclear factor erythroid 2-related factor 2 or HO-1 abrogated the protective effects of AICAR.
CONCLUSIONS
Our data indicate that the primary mechanism for the protective action of AICAR in toxic gas injury is the upregulation of lung HO-1 levels.
Topics: AMP-Activated Protein Kinases; Acute Lung Injury; Aminoimidazole Carboxamide; Animals; Heme Oxygenase-1; Male; Mice; Mice, Inbred C57BL; Rats; Ribonucleotides
PubMed: 34049949
DOI: 10.1183/13993003.03694-2020 -
DNA Repair Jan 2014The incorporation of ribonucleotides in DNA has attracted considerable notice in recent years, since the pool of ribonucleotides can exceed that of the...
The incorporation of ribonucleotides in DNA has attracted considerable notice in recent years, since the pool of ribonucleotides can exceed that of the deoxyribonucleotides by at least 10-20-fold, and single ribonucleotide incorporation by DNA polymerases appears to be a common event. Moreover ribonucleotides are potentially mutagenic and lead to genome instability. As a consequence, errantly incorporated ribonucleotides are rapidly repaired in a process dependent upon RNase H enzymes. On the other hand, global genomic nucleotide excision repair (NER) in prokaryotes and eukaryotes removes damage caused by covalent modifications that typically distort and destabilize DNA through the production of lesions derived from bulky chemical carcinogens, such as polycyclic aromatic hydrocarbon metabolites, or via crosslinking. However, a recent study challenges this lesion-recognition paradigm. The work of Vaisman et al. (2013) [34] reveals that even a single ribonucleotide embedded in a deoxyribonucleotide duplex is recognized by the bacterial NER machinery in vitro. In their report, the authors show that spontaneous mutagenesis promoted by a steric-gate pol V mutant increases in uvrA, uvrB, or uvrC strains lacking rnhB (encoding RNase HII) and to a greater extent in an NER-deficient strain lacking both RNase HI and RNase HII. Using purified UvrA, UvrB, and UvrC proteins in in vitro assays they show that despite causing little distortion, a single ribonucleotide embedded in a DNA duplex is recognized and doubly-incised by the NER complex. We present the hypothesis to explain the recognition and/or verification of this small lesion, that the critical 2'-OH of the ribonucleotide - with its unique electrostatic and hydrogen bonding properties - may act as a signal through interactions with amino acid residues of the prokaryotic NER complex that are not possible with DNA. Such a mechanism might also be relevant if it were demonstrated that the eukaryotic NER machinery likewise incises an embedded ribonucleotide in DNA.
Topics: Binding Sites; DNA; DNA Repair; Genomic Instability; Models, Molecular; Molecular Dynamics Simulation; Ribonuclease H; Ribonucleotides; Substrate Specificity
PubMed: 24290807
DOI: 10.1016/j.dnarep.2013.10.010 -
Proceedings of the National Academy of... Dec 2002Misincorporated ribonucleotides in DNA will cause DNA backbone distortion and may be targeted by DNA repair enzymes. Using double-stranded oligonucleotide probes...
Misincorporated ribonucleotides in DNA will cause DNA backbone distortion and may be targeted by DNA repair enzymes. Using double-stranded oligonucleotide probes containing a single ribose, we demonstrate a robust activity in human, yeast, and Escherichia coli cell-free extracts that nicks 5' of the ribose. The human and yeast extracts also make a subsequent cut 3' of the ribonucleotide releasing a ribonucleotide monophosphate. The resulting 1-nt gap is an ideal substrate for polymerase and ligase to complete a proposed repair sequence that effectively replaces the ribose with deoxyribose. Screening of yeast deletion mutant cells reveals that the initial nick is made by RNase H(35), a RNase H type 2 enzyme, and the second cut is made by Rad27p, the yeast homologue of human FEN-1 protein. RNase H type 2 enzymes are present in all kingdoms of life and are evolutionarily well conserved. We knocked out the corresponding rnhb gene in E. coli and show that extracts from this strain lack the nicking activity. Conversely, a highly purified archaeal RNase HII type 2 protein has a pronounced activity. To study substrate specificity, extracts were made from a yeast double mutant lacking the other main RNase H enzymes [RNase H1 and RNase H(70)], while maintaining RNase H(35). It was found that a single ribose is preferred as substrate over a stretch of riboses, further strengthening a proposed role of this enzyme in the repair of misincorporated ribonucleotides rather than (or in addition to) processing RNADNA hybrid molecules.
Topics: Cell-Free System; DNA; DNA Repair; Endodeoxyribonucleases; Flap Endonucleases; HeLa Cells; Humans; Mutation; Oligonucleotide Probes; Ribonuclease H; Ribonucleotides; Substrate Specificity
PubMed: 12475934
DOI: 10.1073/pnas.262591699 -
Cell Host & Microbe Aug 2018Control of virus infection relies on the stimulation of interferon-stimulated genes (ISGs) that inhibit viral replication. In a recent Nature paper, Gizzi et al. (2018)...
Control of virus infection relies on the stimulation of interferon-stimulated genes (ISGs) that inhibit viral replication. In a recent Nature paper, Gizzi et al. (2018) discovered that the ISG viperin inhibits virus replication by generating the ribonucleotide ddhCTP, which interferes with RNA synthesis, thus offering insights into drug design.
Topics: Antiviral Agents; Genome, Human; Humans; Poisons; Proteins; Ribonucleotides; Virus Replication
PubMed: 30092190
DOI: 10.1016/j.chom.2018.07.014 -
Nucleic Acids Research Nov 2019The presence of ribonucleotides in DNA can lead to genomic instability and cellular lethality. To prevent adventitious rNTP incorporation, the majority of the DNA...
The presence of ribonucleotides in DNA can lead to genomic instability and cellular lethality. To prevent adventitious rNTP incorporation, the majority of the DNA polymerases (dPols) possess a steric filter. The dPol named MsDpo4 (Mycobacterium smegmatis) naturally lacks this steric filter and hence is capable of rNTP addition. The introduction of the steric filter in MsDpo4 did not result in complete abrogation of the ability of this enzyme to incorporate ribonucleotides. In comparison, DNA polymerase IV (PolIV) from Escherichia coli exhibited stringent selection for deoxyribonucleotides. A comparison of MsDpo4 and PolIV led to the discovery of an additional polar filter responsible for sugar selectivity. Thr43 represents the filter in PolIV and this residue forms interactions with the incoming nucleotide to draw it closer to the enzyme surface. As a result, the 2'-OH in rNTPs will clash with the enzyme surface, and therefore ribonucleotides cannot be accommodated in the active site in a conformation compatible with productive catalysis. The substitution of the equivalent residue in MsDpo4-Cys47, with Thr led to a drastic reduction in the ability of the mycobacterial enzyme to incorporate rNTPs. Overall, our studies evince that the polar filter serves to prevent ribonucleotide incorporation by dPols.
Topics: Amino Acid Sequence; Base Sequence; DNA-Directed DNA Polymerase; Kinetics; Models, Molecular; Mycobacterium smegmatis; Ribonucleotides
PubMed: 31544946
DOI: 10.1093/nar/gkz792 -
Journal of the American Chemical Society Aug 2022A growing number of out-of-equilibrium systems have been created and investigated in chemical laboratories over the past decade. One way to achieve this is to create a...
A growing number of out-of-equilibrium systems have been created and investigated in chemical laboratories over the past decade. One way to achieve this is to create a reaction cycle, in which the forward reaction is driven by a chemical fuel and the backward reaction follows a different pathway. Such dissipative reaction networks are still relatively rare, however, and most non-enzymatic examples are based on the carbodiimide-driven generation of carboxylic acid anhydrides. In this work, we describe a dissipative reaction network that comprises the chemically fueled formation of phosphoramidates from natural ribonucleotides (e.g., GMP or AMP) and phosphoramidate hydrolysis as a mild backward reaction. Because the individual reactions are subject to a multitude of interconnected parameters, the software-assisted tool "Design of Experiments" (DoE) was a great asset for optimizing and understanding the network. One notable insight was the stark effect of the nucleophilic catalyst 1-ethylimidazole (EtIm) on the hydrolysis rate, which is reminiscent of the action of the histidine group in phosphoramidase enzymes (e.g., HINT1). We were also able to use the reaction cycle to generate transient self-assemblies, which were characterized by dynamic light scattering (DLS), confocal microscopy (CLSM), and cryogenic transmission electron microscopy (cryo-TEM). Because these compartments are based on prebiotically plausible building blocks, our findings may have relevance for origin-of-life scenarios.
Topics: Amides; Computer-Aided Design; Phosphoric Acids; Ribonucleotides
PubMed: 35953065
DOI: 10.1021/jacs.2c05861 -
PLoS Genetics Feb 2017Previous work has demonstrated the presence of ribonucleotides in human mitochondrial DNA (mtDNA) and in the present study we use a genome-wide approach to precisely map...
Previous work has demonstrated the presence of ribonucleotides in human mitochondrial DNA (mtDNA) and in the present study we use a genome-wide approach to precisely map the location of these. We find that ribonucleotides are distributed evenly between the heavy- and light-strand of mtDNA. The relative levels of incorporated ribonucleotides reflect that DNA polymerase γ discriminates the four ribonucleotides differentially during DNA synthesis. The observed pattern is also dependent on the mitochondrial deoxyribonucleotide (dNTP) pools and disease-causing mutations that change these pools alter both the absolute and relative levels of incorporated ribonucleotides. Our analyses strongly suggest that DNA polymerase γ-dependent incorporation is the main source of ribonucleotides in mtDNA and argues against the existence of a mitochondrial ribonucleotide excision repair pathway in human cells. Furthermore, we clearly demonstrate that when dNTP pools are limiting, ribonucleotides serve as a source of building blocks to maintain DNA replication. Increased levels of embedded ribonucleotides in patient cells with disturbed nucleotide pools may contribute to a pathogenic mechanism that affects mtDNA stability and impair new rounds of mtDNA replication.
Topics: DNA; DNA Polymerase gamma; DNA Repair; DNA Replication; DNA, Mitochondrial; DNA-Directed DNA Polymerase; Fibroblasts; Genome, Mitochondrial; HeLa Cells; Humans; Mitochondria; RNA; Ribonucleases; Ribonucleotides
PubMed: 28207748
DOI: 10.1371/journal.pgen.1006628 -
Biophysical Journal Oct 2017Ribonucleotide incorporation is the most common error occurring during DNA replication. Cells have hence developed mechanisms to remove ribonucleotides from the genome...
Ribonucleotide incorporation is the most common error occurring during DNA replication. Cells have hence developed mechanisms to remove ribonucleotides from the genome and restore its integrity. Indeed, the persistence of ribonucleotides into DNA leads to severe consequences, such as genome instability and replication stress. Thus, it becomes important to understand the effects of ribonucleotides incorporation, starting from their impact on DNA structure and conformation. Here we present a systematic study of the effects of ribonucleotide incorporation into DNA molecules. We have developed, to our knowledge, a new method to efficiently synthesize long DNA molecules (hundreds of basepairs) containing ribonucleotides, which is based on a modified protocol for the polymerase chain reaction. By means of atomic force microscopy, we could therefore investigate the changes, upon ribonucleotide incorporation, of the structural and conformational properties of numerous DNA populations at the single-molecule level. Specifically, we characterized the scaling of the contour length with the number of basepairs and the scaling of the end-to-end distance with the curvilinear distance, the bending angle distribution, and the persistence length. Our results revealed that ribonucleotides affect DNA structure and conformation on scales that go well beyond the typical dimension of the single ribonucleotide. In particular, the presence of ribonucleotides induces a systematic shortening of the molecules, together with a decrease of the persistence length. Such structural changes are also likely to occur in vivo, where they could directly affect the downstream DNA transactions, as well as interfere with protein binding and recognition.
Topics: DNA; Escherichia coli; Linear Models; Microscopy, Atomic Force; Mutation; Nucleic Acid Conformation; Polymerase Chain Reaction; Ribonucleotides; Taq Polymerase
PubMed: 28978432
DOI: 10.1016/j.bpj.2017.07.013