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Nucleic Acids Research Jul 2023The nonenzymatic copying of RNA is thought to have been necessary for the transition between prebiotic chemistry and ribozyme-catalyzed RNA replication in the RNA World....
The nonenzymatic copying of RNA is thought to have been necessary for the transition between prebiotic chemistry and ribozyme-catalyzed RNA replication in the RNA World. We have previously shown that a potentially prebiotic nucleotide activation pathway based on phospho-Passerini chemistry can lead to the efficient synthesis of 2-aminoimidazole activated mononucleotides when carried out under freeze-thaw cycling conditions. Such activated nucleotides react with each other to form 5'-5' 2-aminoimidazolium bridged dinucleotides, enabling template-directed primer extension to occur within the same reaction mixture. However, mononucleotides linked to oligonucleotides by a 5'-5' 2-aminoimidazolium bridge are superior substrates for nonenzymatic primer extension; their higher intrinsic reactivity and their higher template affinity enable faster template copying at lower substrate concentrations. Here we show that eutectic phase phospho-Passerini chemistry efficiently activates short oligonucleotides and promotes the formation of monomer-bridged-oligonucleotide species during freeze-thaw cycles. We then demonstrate that in-situ generated monomer-bridged-oligonucleotides lead to efficient nonenzymatic template copying in the same reaction mixture. Our demonstration that multiple steps in the pathway from activation chemistry to RNA copying can occur together in a single complex environment simplifies this aspect of the origin of life.
Topics: RNA; Oligonucleotides; RNA, Catalytic; Nucleotides; Dinucleoside Phosphates
PubMed: 37247941
DOI: 10.1093/nar/gkad439 -
International Journal of Molecular... Oct 2021DNA lesions that impede fork progression cause replisome stalling and threaten genome stability. RecA, at a lesion-containing gap, interacts with and facilitates DisA...
DNA lesions that impede fork progression cause replisome stalling and threaten genome stability. RecA, at a lesion-containing gap, interacts with and facilitates DisA pausing at these branched intermediates. Paused DisA suppresses its synthesis of the essential c-di-AMP messenger. The RuvAB-RecU resolvasome branch migrates and resolves formed Holliday junctions (HJ). We show that DisA prevents DNA degradation. DisA, which interacts with RuvB, binds branched structures, and reduces the RuvAB DNA-dependent ATPase activity. DisA pre-bound to HJ DNA limits RuvAB and RecU activities, but such inhibition does not occur if the RuvAB- or RecU-HJ DNA complexes are pre-formed. RuvAB or RecU pre-bound to HJ DNA strongly inhibits DisA-mediated synthesis of c-di-AMP, and indirectly blocks cell proliferation. We propose that DisA limits RuvAB-mediated fork remodeling and RecU-mediated HJ cleavage to provide time for damage removal and replication restart in order to preserve genome integrity.
Topics: Adenosine Triphosphatases; Bacillus subtilis; Bacterial Proteins; Chromosome Breakage; DNA Helicases; DNA Replication; DNA, Bacterial; DNA, Cruciform; DNA-Binding Proteins; Dinucleoside Phosphates; Escherichia coli; Holliday Junction Resolvases; Magnesium; Phosphorus-Oxygen Lyases
PubMed: 34768753
DOI: 10.3390/ijms222111323 -
MBio Feb 2021In Bacillus subtilis and other Gram-positive bacteria, cyclic di-AMP is an essential second messenger that signals potassium availability by binding to a variety of...
In Bacillus subtilis and other Gram-positive bacteria, cyclic di-AMP is an essential second messenger that signals potassium availability by binding to a variety of proteins. In some bacteria, c-di-AMP also binds to the pyruvate carboxylase to inhibit its activity. We have discovered that in B. subtilis the c-di-AMP target protein DarB, rather than c-di-AMP itself, specifically binds to pyruvate carboxylase both and . This interaction stimulates the activity of the enzyme, as demonstrated by enzyme assays and metabolite determinations. Both the interaction and the activation of enzyme activity require apo-DarB and are inhibited by c-di-AMP. Under conditions of potassium starvation and corresponding low c-di-AMP levels, the demand for citric acid cycle intermediates is increased. Apo-DarB helps to replenish the cycle by activating both pyruvate carboxylase gene expression and enzymatic activity via triggering the stringent response as a result of its interaction with the (p)ppGpp synthetase Rel and by direct interaction with the enzyme, respectively. If bacteria experience a starvation for potassium, by far the most abundant metal ion in every living cell, they have to activate high-affinity potassium transporters, switch off growth activities such as translation and transcription of many genes or replication, and redirect the metabolism in a way that the most essential functions of potassium can be taken over by metabolites. Importantly, potassium starvation triggers a need for glutamate-derived amino acids. In many bacteria, the responses to changing potassium availability are orchestrated by a nucleotide second messenger, cyclic di-AMP. c-di-AMP binds to factors involved directly in potassium homeostasis and to dedicated signal transduction proteins. Here, we demonstrate that in the Gram-positive model organism Bacillus subtilis, the c-di-AMP receptor protein DarB can bind to and, thus, activate pyruvate carboxylase, the enzyme responsible for replenishing the citric acid cycle. This interaction takes place under conditions of potassium starvation if DarB is present in the apo form and the cells are in need of glutamate. Thus, DarB links potassium availability to the control of central metabolism.
Topics: Cyclic AMP; Bacillus subtilis; Pyruvate Carboxylase; Bacterial Proteins; Second Messenger Systems; Dinucleoside Phosphates; Glutamic Acid; Potassium
PubMed: 35130724
DOI: 10.1128/mbio.03602-21 -
Nature Immunology Feb 2022
Topics: Anions; Dinucleoside Phosphates; T-Lymphocytes
PubMed: 35105986
DOI: 10.1038/s41590-021-01118-6 -
Journal of Bacteriology Sep 20213'3'-Cyclic di-AMP (c-di-AMP) is an important nucleotide second messenger found throughout the bacterial domain of life. c-di-AMP is essential in many bacteria and...
3'3'-Cyclic di-AMP (c-di-AMP) is an important nucleotide second messenger found throughout the bacterial domain of life. c-di-AMP is essential in many bacteria and regulates a diverse array of effector proteins controlling pathogenesis, cell wall homeostasis, osmoregulation, and central metabolism. Despite the ubiquity and importance of c-di-AMP, methods to detect this signaling molecule are limited, particularly at single-cell resolution. In this work, crystallization of the Listeria monocytogenes c-di-AMP effector protein Lmo0553 enabled structure-guided design of a Förster resonance energy transfer (FRET)-based biosensor, which we have named CDA5. CDA5 is a fully genetically encodable, specific, and reversible biosensor which allows the detection of c-di-AMP dynamics both and within live cells in a nondestructive manner. Our initial studies identified a distribution of c-di-AMP in Bacillus subtilis populations first grown in Luria broth and then resuspended in diluted Luria broth compatible with fluorescence analysis. Furthermore, we found that B. subtilis mutants lacking either a c-di-AMP phosphodiesterase and cyclase have higher and lower FRET responses, respectively. These findings provide novel insight into the c-di-AMP distribution within bacterial populations and establish CDA5 as a powerful platform for characterizing new aspects of c-di-AMP regulation. c-di-AMP is an important nucleotide second messenger for which detection methods are severely limited. In this work we engineered and implemented a c-di-AMP-specific FRET biosensor to remedy this dearth. We present this biosensor, CDA5, as a versatile tool to investigate previously intractable facets of c-di-AMP biology.
Topics: Bacillus subtilis; Bacterial Proteins; Biosensing Techniques; Dinucleoside Phosphates; Fluorescence Resonance Energy Transfer; Gene Expression Regulation, Bacterial; Listeria monocytogenes; Models, Molecular; Mutation; Nucleotides; Protein Conformation
PubMed: 34309402
DOI: 10.1128/JB.00080-21 -
MBio Aug 2020The facultative intracellular pathogen , like many related , uses the nucleotide second messenger cyclic di-AMP (c-di-AMP) to adapt to changes in nutrient availability,...
The facultative intracellular pathogen , like many related , uses the nucleotide second messenger cyclic di-AMP (c-di-AMP) to adapt to changes in nutrient availability, osmotic stress, and the presence of cell wall-acting antibiotics. In rich medium, c-di-AMP is essential; however, mutations in , the gene encoding c-di-AMP binding protein B, suppress essentiality. In this study, we identified that the reason for -dependent essentiality is through induction of the stringent response by RelA. RelA is a bifunctional RelA/SpoT homolog (RSH) that modulates levels of (p)ppGpp, a secondary messenger that orchestrates the stringent response through multiple allosteric interactions. We performed a forward genetic suppressor screen on bacteria lacking c-di-AMP to identify genomic mutations that rescued growth while was constitutively expressed and identified mutations in the synthetase domain of RelA. The synthetase domain of RelA was also identified as an interacting partner of CbpB in a yeast-2-hybrid screen. Biochemical analyses confirmed that free CbpB activates RelA while c-di-AMP inhibits its activation. We solved the crystal structure of CbpB bound and unbound to c-di-AMP and provide insight into the region important for c-di-AMP binding and RelA activation. The results of this study show that CbpB completes a homeostatic regulatory circuit between c-di-AMP and (p)ppGpp in Bacteria must efficiently maintain homeostasis of essential molecules to survive in the environment. We found that the levels of c-di-AMP and (p)ppGpp, two nucleotide second messengers that are highly conserved throughout the microbial world, coexist in a homeostatic loop in the facultative intracellular pathogen Here, we found that cyclic di-AMP binding protein B (CbpB) acts as a c-di-AMP sensor that promotes the synthesis of (p)ppGpp by binding to RelA when c-di-AMP levels are low. Addition of c-di-AMP prevented RelA activation by binding and sequestering CbpB. Previous studies showed that (p)ppGpp binds and inhibits c-di-AMP phosphodiesterases, resulting in an increase in c-di-AMP. This pathway is controlled via direct enzymatic regulation and indicates an additional mechanism of ribosome-independent stringent activation.
Topics: Animals; Bacterial Proteins; Dinucleoside Phosphates; Gene Expression Regulation, Bacterial; Guanosine Pentaphosphate; Homeostasis; Listeria monocytogenes; Mice; Protein Binding; Second Messenger Systems; Signal Transduction
PubMed: 32843560
DOI: 10.1128/mBio.01625-20 -
Chemistry (Weinheim An Der Bergstrasse,... May 2024Biomolecules containing adenosine di- or triphosphate (ADP or ATP) are crucial for diverse biological processes. Synthesis of these biomolecules and development of their...
Biomolecules containing adenosine di- or triphosphate (ADP or ATP) are crucial for diverse biological processes. Synthesis of these biomolecules and development of their chemical probes are important to elucidate their functions. Enabling reproducible and high-yielding access to these ADP- and ATP-containing molecules via conventional P(III)-P(V) and P(V)-P(V) coupling reactions is challenging owing to water content in highly polar phosphate-containing substrates. Herein, we report an efficient and reliable method for protecting-group-free P(V)-P(V) coupling reaction through in situ activation of phosphates using hydrolysis-stable 2-[N-(2-methylimidazoyl)]-1,3-dimethylimidazolinium chloride (2-MeImIm-Cl), providing the corresponding electrophilic P(V) intermediates for subsequent nucleophilic attack using their coupling partners. This P(V)-P(V) coupling reaction proceeded even in a wet reaction medium and showed a broad substrate scope, accommodating protecting-group-free synthesis of ADP-ribose and nicotinamide adenine diphosphate analogs, ATP-containing biomolecules, and ADP-ribosyl peptides.
PubMed: 38763895
DOI: 10.1002/chem.202401302 -
Proceedings of the National Academy of... Apr 2024In bacteria, intracellular K is involved in the regulation of membrane potential, cytosolic pH, and cell turgor as well as in spore germination, environmental...
In bacteria, intracellular K is involved in the regulation of membrane potential, cytosolic pH, and cell turgor as well as in spore germination, environmental adaptation, cell-to-cell communication in biofilms, antibiotic sensitivity, and infectivity. The second messenger cyclic-di-AMP (c-di-AMP) has a central role in modulating the intracellular K concentration in many bacterial species, controlling transcription and function of K channels and transporters. However, our understanding of how this regulatory network responds to c-di-AMP remains poor. We used the RCK (Regulator of Conductance of K) proteins that control the activity of Ktr channels in as a model system to analyze the regulatory function of c-di-AMP with a combination of in vivo and in vitro functional and structural characterization. We determined that the two RCK proteins (KtrA and KtrC) are neither physiologically redundant or functionally equivalent. KtrC is the physiologically dominant RCK protein in the regulation of Ktr channel activity. In explaining this hierarchical organization, we found that, unlike KtrA, KtrC is very sensitive to c-di-AMP inactivation and lack of c-di-AMP regulation results in RCK protein toxicity, most likely due to unregulated K flux. We also found that KtrC can assemble with KtrA, conferring c-di-AMP regulation to the functional KtrA/KtrC heteromers and potentially compensating KtrA toxicity. Altogether, we propose that the central role of c-di-AMP in the control of the K machinery, by modulating protein levels through gene transcription and by regulating protein activity, has determined the evolutionary selection of KtrC as the dominant RCK protein, shaping the hierarchical organization of regulatory components of the K machinery.
Topics: Bacterial Proteins; Bacillus subtilis; Potassium; Gene Expression Regulation, Bacterial; Dinucleoside Phosphates; Potassium Channels
PubMed: 38652747
DOI: 10.1073/pnas.2318666121 -
International Journal of Biological... 2022Chronic Hepatitis B virus (CHB) infection is a global public health problem. Oligodeoxynucleotides (ODNs) containing class C unmethylated cytosine-guanine dinucleotide...
Chronic Hepatitis B virus (CHB) infection is a global public health problem. Oligodeoxynucleotides (ODNs) containing class C unmethylated cytosine-guanine dinucleotide (CpG-C) motifs may provide potential adjuvants for the immunotherapeutic strategy against CHB, since CpG-C ODNs stimulate both B cell and dendritic cell (DC) activation. However, the efficacy of CpG-C ODN as an anti-HBV vaccine adjuvant remains unclear. In this study, we demonstrated that CpG M362 (CpG-C ODN) as an adjuvant in anti-HBV vaccine (cHBV-vaccine) successfully and safely eliminated the virus in HBV-carrier mice. The cHBV-vaccine enhanced DC maturation both and , overcame immune tolerance, and recovered exhausted T cells in HBV-carrier mice. Furthermore, the cHBV-vaccine elicited robust hepatic HBV-specific CD8 and CD4 T cell responses, with increased cellular proliferation and IFN-γ secretion. Additionally, the cHBV-vaccine invoked a long-lasting follicular CXCR5 CD8 T cell response following HBV re-challenge. Taken together, CpG M362 in combination with rHBVvac cleared persistent HBV and achieved long-term virological control, making it a promising candidate for treating CHB.
Topics: Adjuvants, Immunologic; Animals; Dinucleoside Phosphates; Disease Models, Animal; Hepatitis B Vaccines; Hepatitis B, Chronic; Male; Mice; Mice, Inbred C57BL; Oligodeoxyribonucleotides
PubMed: 34975324
DOI: 10.7150/ijbs.62424 -
Biochemistry Feb 2021Aminoacylated tRNAs are the substrates for ribosomal protein synthesis in all branches of life, implying an ancient origin for aminoacylation chemistry. In the 1970s,...
Aminoacylated tRNAs are the substrates for ribosomal protein synthesis in all branches of life, implying an ancient origin for aminoacylation chemistry. In the 1970s, Orgel and colleagues reported potentially prebiotic routes to aminoacylated nucleotides and their RNA-templated condensation to form amino acid-bridged dinucleotides. However, it is unclear whether such reactions would have aided or impeded non-enzymatic RNA replication. Determining whether aminoacylated RNAs could have been advantageous in evolution prior to the emergence of protein synthesis remains a key challenge. We therefore tested the ability of aminoacylated RNA to participate in both templated primer extension and ligation reactions. We find that at low magnesium concentrations that favor fatty acid-based protocells, these reactions proceed orders of magnitude more rapidly than when initiated from the -diol of unmodified RNA. We further demonstrate that amino acid-bridged RNAs can act as templates in a subsequent round of copying. Our results suggest that aminoacylation facilitated non-enzymatic RNA replication, thus outlining a potentially primordial functional link between aminoacylation chemistry and RNA replication.
Topics: Aminoacylation; Dinucleoside Phosphates; Nucleic Acid Conformation; Nucleotides; RNA; Templates, Genetic; Transfer RNA Aminoacylation
PubMed: 33523633
DOI: 10.1021/acs.biochem.0c00943