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The EMBO Journal Sep 2023The building blocks for RNA and DNA are made in the cytosol, meaning mitochondria depend on the import and salvage of ribonucleoside triphosphates (rNTPs) and...
The building blocks for RNA and DNA are made in the cytosol, meaning mitochondria depend on the import and salvage of ribonucleoside triphosphates (rNTPs) and deoxyribonucleoside triphosphates (dNTPs) for the synthesis of their own genetic material. While extensive research has focused on mitochondrial dNTP homeostasis due to its defects being associated with various mitochondrial DNA (mtDNA) depletion and deletion syndromes, the investigation of mitochondrial rNTP homeostasis has received relatively little attention. In this issue of the EMBO Journal, Grotehans et al provide compelling evidence of a major role for NME6, a mitochondrial nucleoside diphosphate kinase, in the conversion of pyrimidine ribonucleoside diphosphates into the corresponding triphosphates. These data also suggest a significant physiological role for NME6, as its absence results in the depletion of mitochondrial transcripts and destabilization of the electron transport chain (Grotehans et al, 2023).
Topics: Ribonucleotides; Mitochondria; DNA, Mitochondrial; Nucleotides; Ribonucleosides
PubMed: 37548337
DOI: 10.15252/embj.2023114990 -
Biochemistry Sep 2023Because purine nucleotides are essential for all life, differences between how microbes and humans metabolize purines can be exploited for the development of...
Because purine nucleotides are essential for all life, differences between how microbes and humans metabolize purines can be exploited for the development of antimicrobial therapies. While humans biosynthesize purine nucleotides in a 10-step pathway, most microbes utilize an additional 11th enzymatic activity. The human enzyme, aminoimidazole ribonucleotide (AIR) carboxylase generates the product 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) directly. Most microbes, however, require two separate enzymes, a synthetase (PurK) and a mutase (PurE), and proceed through the intermediate, N-CAIR. Toward the development of therapeutics that target these differences, we have solved crystal structures of the N-CAIR mutase of the human pathogens (LpPurE) and (BcPurE) and used a structure-guided approach to identify inhibitors. Analysis of the structures reveals a highly conserved fold and active site architecture. Using this data, and three additional structures of PurE enzymes, we screened a library of FDA-approved compounds and identified a set of 25 candidates for further analysis. Among these, we identified several new PurE inhibitors with micromolar IC values. Several of these compounds, including the α-blocker Alfuzosin, inhibit the microbial PurE enzymes much more effectively than the human homologue. These structures and the newly described PurE inhibitors are valuable tools to aid in further studies of this enzyme and provide a foundation for the development of compounds that target differences between human and microbial purine metabolism.
Topics: Humans; Ribonucleotides; Escherichia coli; Intramolecular Transferases; Purine Nucleotides
PubMed: 37552766
DOI: 10.1021/acs.biochem.2c00705 -
International Journal of Molecular... Mar 2020In the last decade, it has become evident that RNA is frequently found in DNA. It is now well established that single embedded ribonucleoside monophosphates (rNMPs) are... (Review)
Review
In the last decade, it has become evident that RNA is frequently found in DNA. It is now well established that single embedded ribonucleoside monophosphates (rNMPs) are primarily introduced by DNA polymerases and that longer stretches of RNA can anneal to DNA, generating RNA:DNA hybrids. Among them, the most studied are R-loops, peculiar three-stranded nucleic acid structures formed upon the re-hybridization of a transcript to its template DNA. In addition, polyribonucleotide chains are synthesized to allow DNA replication priming, double-strand breaks repair, and may as well result from the direct incorporation of consecutive rNMPs by DNA polymerases. The bright side of RNA into DNA is that it contributes to regulating different physiological functions. The dark side, however, is that persistent RNA compromises genome integrity and genome stability. For these reasons, the characterization of all these structures has been under growing investigation. In this review, we discussed the origin of single and multiple ribonucleotides in the genome and in the DNA of organelles, focusing on situations where the aberrant processing of RNA:DNA hybrids may result in multiple rNMPs embedded in DNA. We concluded by providing an overview of the currently available strategies to study the presence of single and multiple ribonucleotides in DNA in vivo.
Topics: Animals; DNA; DNA Replication; Genomic Instability; Humans; Nucleic Acid Heteroduplexes; R-Loop Structures; Ribonucleotides
PubMed: 32131532
DOI: 10.3390/ijms21051706 -
Annual Review of Biochemistry Jun 2022Our current view of how DNA-based genomes are efficiently and accurately replicated continues to evolve as new details emerge on the presence of ribonucleotides in DNA.... (Review)
Review
Our current view of how DNA-based genomes are efficiently and accurately replicated continues to evolve as new details emerge on the presence of ribonucleotides in DNA. Ribonucleotides are incorporated during eukaryotic DNA replication at rates that make them the most common noncanonical nucleotide placed into the nuclear genome, they are efficiently repaired, and their removal impacts genome integrity. This review focuses on three aspects of this subject: the incorporation of ribonucleotides into the eukaryotic nuclear genome during replication by B-family DNA replicases, how these ribonucleotides are removed, and the consequences of their presence or removal for genome stability and disease.
Topics: DNA; DNA Repair; DNA Replication; Eukaryota; Genomic Instability; Nucleotidyltransferases; Ribonucleotides
PubMed: 35287470
DOI: 10.1146/annurev-biochem-032620-110354 -
The EMBO Journal Sep 2023Replication of the mitochondrial genome and expression of the genes it encodes both depend on a sufficient supply of nucleotides to mitochondria. Accordingly,...
Replication of the mitochondrial genome and expression of the genes it encodes both depend on a sufficient supply of nucleotides to mitochondria. Accordingly, dysregulated nucleotide metabolism not only destabilises the mitochondrial genome, but also affects its transcription. Here, we report that a mitochondrial nucleoside diphosphate kinase, NME6, supplies mitochondria with pyrimidine ribonucleotides that are necessary for the transcription of mitochondrial genes. Loss of NME6 function leads to the depletion of mitochondrial transcripts, as well as destabilisation of the electron transport chain and impaired oxidative phosphorylation. These deficiencies are rescued by an exogenous supply of pyrimidine ribonucleosides. Moreover, NME6 is required for the maintenance of mitochondrial DNA when the access to cytosolic pyrimidine deoxyribonucleotides is limited. Our results therefore reveal an important role for ribonucleotide salvage in mitochondrial gene expression.
Topics: Genes, Mitochondrial; Pyrimidines; Mitochondria; Nucleotides; DNA, Mitochondrial; Ribonucleotides
PubMed: 37439264
DOI: 10.15252/embj.2022113256 -
Critical Reviews in Biochemistry and... Feb 2021Ribonucleotides are the most abundant non-canonical nucleotides in the genome. Their vast presence and influence over genome biology is becoming increasingly... (Review)
Review
Ribonucleotides are the most abundant non-canonical nucleotides in the genome. Their vast presence and influence over genome biology is becoming increasingly appreciated. Here we review the recent progress made in understanding their genomic presence, incorporation characteristics and usefulness as biomarkers for polymerase enzymology. We also discuss ribonucleotide processing, the genetic consequences of unrepaired ribonucleotides in DNA and evidence supporting the significance of their transient presence in the nuclear genome.
Topics: Animals; Biomarkers; Cell Nucleus; DNA; DNA Repair; DNA Replication; DNA-Directed DNA Polymerase; Genome, Mitochondrial; Genomic Instability; Humans; Ribonucleotides; Saccharomyces cerevisiae
PubMed: 33461360
DOI: 10.1080/10409238.2020.1869175 -
Critical Reviews in Biochemistry and... 2015In all living cells, DNA is the storage medium for genetic information. Being quite stable, DNA is well-suited for its role in storage and propagation of information,... (Review)
Review
In all living cells, DNA is the storage medium for genetic information. Being quite stable, DNA is well-suited for its role in storage and propagation of information, but RNA is also covalently included in DNA through various mechanisms. Recent studies also demonstrate useful aspects of including ribonucleotides in the genome during repair. Therefore, our understanding of the consequences of RNA inclusion into bacterial genomic DNA is just beginning, but with its high frequency of occurrence the consequences and potential benefits are likely to be numerous and diverse. In this review, we discuss the processes that cause ribonucleotide inclusion in genomic DNA, the pathways important for ribonucleotide removal and the consequences that arise should ribonucleotides remain nested in genomic DNA.
Topics: Bacillus subtilis; DNA Polymerase I; DNA Repair; DNA Replication; DNA, Bacterial; Escherichia coli; Ribonucleotides
PubMed: 25387798
DOI: 10.3109/10409238.2014.981647 -
Molecules (Basel, Switzerland) Apr 2022A diastereoselective synthesis of the β-anomer of glycinamide ribonucleotide (β-GAR) has been developed. The synthesis was accomplished in nine steps from D-ribose and...
A diastereoselective synthesis of the β-anomer of glycinamide ribonucleotide (β-GAR) has been developed. The synthesis was accomplished in nine steps from D-ribose and occurred in 5% overall yield. The route provided material on the multi-milligram scale. The synthetic β-GAR formed was remarkably resistant to anomerization both in solution and as a solid.
Topics: Glycine; Hydroxymethyl and Formyl Transferases; Phosphoribosylglycinamide Formyltransferase; Ribonucleotides
PubMed: 35458726
DOI: 10.3390/molecules27082528 -
Nature Communications Jun 2019Ribonucleotide reductases (RNRs) use a conserved radical-based mechanism to catalyze the conversion of ribonucleotides to deoxyribonucleotides. Within the RNR family,...
Ribonucleotide reductases (RNRs) use a conserved radical-based mechanism to catalyze the conversion of ribonucleotides to deoxyribonucleotides. Within the RNR family, class Ib RNRs are notable for being largely restricted to bacteria, including many pathogens, and for lacking an evolutionarily mobile ATP-cone domain that allosterically controls overall activity. In this study, we report the emergence of a distinct and unexpected mechanism of activity regulation in the sole RNR of the model organism Bacillus subtilis. Using a hypothesis-driven structural approach that combines the strengths of small-angle X-ray scattering (SAXS), crystallography, and cryo-electron microscopy (cryo-EM), we describe the reversible interconversion of six unique structures, including a flexible active tetramer and two inhibited helical filaments. These structures reveal the conformational gymnastics necessary for RNR activity and the molecular basis for its control via an evolutionarily convergent form of allostery.
Topics: Allosteric Regulation; Allosteric Site; Bacillus subtilis; Bacterial Proteins; Cryoelectron Microscopy; Crystallography, X-Ray; Evolution, Molecular; Models, Molecular; Protein Structure, Quaternary; Ribonucleotide Reductases; Ribonucleotides; Scattering, Small Angle
PubMed: 31201319
DOI: 10.1038/s41467-019-10568-4 -
Critical Reviews in Biochemistry and... Aug 2018The well-being of all living organisms relies on the accurate duplication of their genomes. This is usually achieved by highly elaborate replicase complexes which ensure... (Review)
Review
The well-being of all living organisms relies on the accurate duplication of their genomes. This is usually achieved by highly elaborate replicase complexes which ensure that this task is accomplished timely and efficiently. However, cells often must resort to the help of various additional "specialized" DNA polymerases that gain access to genomic DNA when replication fork progression is hindered. One such specialized polymerase family consists of the so-called "translesion synthesis" (TLS) polymerases; enzymes that have evolved to replicate damaged DNA. To fulfill their main cellular mission, TLS polymerases often must sacrifice precision when selecting nucleotide substrates. Low base-substitution fidelity is a well-documented inherent property of these enzymes. However, incorrect nucleotide substrates are not only those which do not comply with Watson-Crick base complementarity, but also those whose sugar moiety is incorrect. Does relaxed base-selectivity automatically mean that the TLS polymerases are unable to efficiently discriminate between ribonucleoside triphosphates and deoxyribonucleoside triphosphates that differ by only a single atom? Which strategies do TLS polymerases employ to select suitable nucleotide substrates? In this review, we will collate and summarize data accumulated over the past decade from biochemical and structural studies, which aim to answer these questions.
Topics: DNA; DNA-Directed DNA Polymerase; Ribonucleotides
PubMed: 29972306
DOI: 10.1080/10409238.2018.1483889