RESUMO
The linear chromosome of Streptomyces exhibits a highly compartmentalized structure with a conserved central region flanked by variable arms. As double strand break (DSB) repair mechanisms play a crucial role in shaping the genome plasticity of Streptomyces, we investigated the role of EndoMS/NucS, a recently characterized endonuclease involved in a non-canonical mismatch repair (MMR) mechanism in archaea and actinobacteria, that singularly corrects mismatches by creating a DSB. We showed that Streptomyces mutants lacking NucS display a marked colonial phenotype and a drastic increase in spontaneous mutation rate. In vitro biochemical assays revealed that NucS cooperates with the replication clamp to efficiently cleave G/T, G/G and T/T mismatched DNA by producing DSBs. These findings are consistent with the transition-shifted mutational spectrum observed in the mutant strains and reveal that NucS-dependent MMR specific task is to eliminate G/T mismatches generated by the DNA polymerase during replication. Interestingly, our data unveil a crescent-shaped distribution of the transition frequency from the replication origin towards the chromosomal ends, shedding light on a possible link between NucS-mediated DSBs and Streptomyces genome evolution.
Assuntos
Cromossomos Bacterianos , Reparo de Erro de Pareamento de DNA , Endonucleases , Streptomyces , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Pareamento Incorreto de Bases , Cromossomos Bacterianos/genética , Quebras de DNA de Cadeia Dupla , Reparo de Erro de Pareamento de DNA/genética , Replicação do DNA/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/genética , Endonucleases/genética , Endonucleases/metabolismo , Mutação , Taxa de Mutação , Streptomyces/genética , Streptomyces/enzimologiaRESUMO
Asgard archaea include the closest known archaeal relatives of eukaryotes. Here, we investigate the evolution and function of Asgard thymidylate synthases and other folate-dependent enzymes required for the biosynthesis of DNA, RNA, amino acids and vitamins, as well as syntrophic amino acid utilization. Phylogenies of Asgard folate-dependent enzymes are consistent with their horizontal transmission from various bacterial groups. We experimentally validate the functionality of thymidylate synthase ThyX of the cultured 'Candidatus Prometheoarchaeum syntrophicum'. The enzyme efficiently uses bacterial-like folates and is inhibited by mycobacterial ThyX inhibitors, even though the majority of experimentally tested archaea are known to use carbon carriers distinct from bacterial folates. Our phylogenetic analyses suggest that the eukaryotic thymidylate synthase, required for de novo DNA synthesis, is not closely related to archaeal enzymes and might have been transferred from bacteria to protoeukaryotes during eukaryogenesis. Altogether, our study suggests that the capacity of eukaryotic cells to duplicate their genetic material is a sum of archaeal (replisome) and bacterial (thymidylate synthase) characteristics. We also propose that recent prevalent lateral gene transfer from bacteria has markedly shaped the metabolism of Asgard archaea.
Assuntos
Archaea , Eucariotos , Archaea/metabolismo , Eucariotos/genética , Eucariotos/metabolismo , Filogenia , Timidilato Sintase/genética , Timidilato Sintase/metabolismo , Bactérias/genética , Bactérias/metabolismo , Aminoácidos/metabolismo , Ácido Fólico/metabolismo , DNA/metabolismoRESUMO
In 2002, a new class of thymidylate synthase (TS) involved in the de novo synthesis of dTMP named Flavin-Dependent Thymidylate Synthase (FDTS) encoded by the thyX gene was discovered; FDTS is present only in 30% of prokaryote pathogens and not in human pathogens, which makes it an attractive target for the development of new antibacterial agents, especially against multi-resistant pathogens. We report herein the synthesis and structure-activity relationship of a novel series of hitherto unknown pyrido[1,2-e]purine-2,4(1H,3H)-dione analogues. Several synthetics efforts were done to optimize regioselective N1-alkylation through organopalladium cross-coupling. Modelling of potential hits were performed to generate a model of interaction into the active pocket of FDTS to understand and guide further synthetic modification. All those compounds were evaluated on an in-house in vitro NADPH oxidase assays screening as well as against Mycobacterium tuberculosis ThyX. The highest inhibition was obtained for compound 23a with 84.3% at 200 µM without significant cytotoxicity (CC50 > 100 µM) on PBM cells.
Assuntos
Mycobacterium tuberculosis , Antibacterianos/farmacologia , Dinitrocresóis , Flavinas/metabolismo , Flavinas/farmacologia , Humanos , Mycobacterium tuberculosis/genética , NADPH Oxidases , Purinas/farmacologia , Relação Estrutura-Atividade , Timidina Monofosfato , Timidilato Sintase/metabolismoRESUMO
The labeling and specific detection of nascent DNA by the incorporation of thymidine analogs provide crucial information about DNA replication dynamics without requiring the intracellular expression of fluorescent proteins. After cell fixation and permeabilization, specific detection of thymidine analogs by antibodies can be performed using super-resolution imaging techniques. Here we describe a protocol to label nascent DNA using 5'-bromo-2'-deoxyuridine (BrdU) in Haloferax volcanii cells and generate super-resolved images of neo-synthesized DNA foci either by 3D Structured illumination microscopy (3D-SIM) or Stochastic Optical Reconstruction Microscopy (STORM).
Assuntos
Haloferax volcanii , Microscopia , Bromodesoxiuridina , DNA , Microscopia/métodos , TimidinaRESUMO
Flavin-Dependent Thymidylate Synthase (FDTS) encoded by ThyX gene was discovered as a new class of thymidylate synthase involved in the de novo synthesis of dTMP named only in 30 % of human pathogenic bacteria. This target was pursed for the development of new antibacterial agents against multiresistant pathogens. We have developed a new class of ANPs based on the mimic of two natural's cofactors (dUMP and FAD) as inhibitors against Mycobacterium tuberculosis ThyX. Several synthetic efforts were performed to optimize regioselective N1-alkylation, cross-coupling metathesis and Sonogashira cross-coupling. Compound 19c showed a poor 31.8% inhibitory effect on ThyX at 200 µM.
Assuntos
Antibacterianos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Nucleosídeos/farmacologia , Timidilato Sintase/antagonistas & inibidores , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mycobacterium tuberculosis/enzimologia , Nucleosídeos/síntese química , Nucleosídeos/química , Relação Estrutura-Atividade , Timidilato Sintase/metabolismoRESUMO
Quenching of flavin fluorescence by electron transfer from neighboring aromatic residues is ubiquitous in flavoproteins. Apart from constituting a functional process in specific light-active systems, time-resolved spectral characterization of the process can more generally be employed as a probe for the active site configuration and dynamics. In the C51A variant of the bacterial RNA-transforming flavoenzyme TrmFO from the bacterium Thermus thermophilus, fluorescence is very short-lived (~ 1 ps), and close-by Tyr343 is known to act as the main quencher, as confirmed here by the very similar dynamics observed in protein variants with modified other potential quenchers, Trp283 and Trp214. When Tyr343 is modified to redox-inactive phenylalanine, slower and highly multiphasic kinetics are observed on the picosecond-nanosecond timescale, reflecting heterogeneous electron donor-acceptor configurations. We demonstrate that Trp214, which is located on a potentially functional flexible loop, contributes to electron donor quenching in this variant. Contrasting with observations in other nucleic acid-transforming enzymes, these kinetics are strikingly temperature-independent. This indicates (a) near-barrierless electron transfer reactions and (b) no exchange between different configurations on the timescale up to at least 2 ns, despite the presumed flexibility of Trp214. Results of extensive molecular dynamics simulations are presented to explain this unexpected finding in terms of slowly exchanging protein configurations.
Assuntos
Proteínas de Bactérias/metabolismo , Simulação de Dinâmica Molecular , Thermus thermophilus/enzimologia , Proteínas de Bactérias/química , Sítios de Ligação , Proteínas de Ligação ao GTP , Processos FotoquímicosRESUMO
Cyclodipeptide synthases (CDPSs) perform nonribosomal protein synthesis using two aminoacyl-tRNA substrates to produce cyclodipeptides. At present, there are no structural details of the CDPS:tRNA interaction available. Using AlbC, a CDPS that produces cyclo(l-Phe-l-Phe), the interaction between AlbC and its Phe-tRNA substrate was investigated. Simulations of models of the AlbC:tRNA complex, proposed by rigid-body docking or homology modeling, demonstrated that interactions with residues of an AlbC α-helix, α4, significantly contribute to the free energy of binding of AlbC to tRNA. Individual residue contributions to the tRNA binding free energy of the discovered binding mode explain well the available biochemical data, and the results of in vivo assay experiments performed in this work and guided by simulations. In molecular dynamics simulations, the phenylalanyl group predominantly occupied the two positions observed in the experimental structure of AlbC in the dipeptide intermediate state, suggesting that tRNAs of the first and second substrates interact with AlbC in a similar manner. Overall, given the high degree of sequence and structural similarity among the members of the CDPS NYH protein subfamily, the mechanism of the protein:tRNA interaction is expected to be pertinent to a wide range of proteins interacting with tRNA.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Peptídeo Sintases/química , Peptídeo Sintases/metabolismo , Peptídeos Cíclicos/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Streptomyces/metabolismo , Proteínas de Bactérias/genética , Modelos Moleculares , Mutação , Peptídeo Sintases/genética , Peptídeos Cíclicos/química , Conformação Proteica em alfa-Hélice , Aminoacil-RNA de Transferência/químicaRESUMO
Naphthoquinones (NQs) are natural and synthetic compounds with a wide range of biological activities commonly attributed to their redox activity and/or chemical reactivity. However, genetic and biochemical experiments have recently demonstrated that 2-hydroxy-NQs (2-OH-NQs) act as highly specific noncovalent inhibitors of the essential bacterial thymidylate synthase ThyX in a cellular context. We used biochemical experiments and molecular dynamics simulations to elucidate the selective inhibition mechanism of NQ inhibitors of ThyX from Mycobacterium tuberculosis (Mtb). Free energy simulations rationalized how ThyX recognizes the natural substrate dUMP in the N3-ionized form using an arginine, Arg199, in Mtb. The results further demonstrated that 2-OH-NQ, similar to dUMP, binds to ThyX in the ionized form, and the strong and selective binding of 2-OH-NQ to ThyX is also explained by electrostatic interactions with Arg199. The stronger binding of the close analog 5F-dUMP to ThyX and its inhibitory properties compared with dUMP were explained by the stronger acidity of the uracil N3 atom. Our results, therefore, revealed that the ionization of 2-OH-NQs drives their biological activities by mimicking the interactions with the natural substrate. Our observations encourage the rational design of optimized ThyX inhibitors that ultimately may serve as antibiotics.
Assuntos
Mycobacterium tuberculosis , Naftoquinonas , Simulação de Dinâmica Molecular , Mycobacterium tuberculosis/metabolismo , Naftoquinonas/farmacologia , Timidilato Sintase/metabolismoRESUMO
The importance of unusual DNA structures in the regulation of basic cellular processes is an emerging field of research. Amongst local non-B DNA structures, G-quadruplexes (G4s) have gained in popularity during the last decade, and their presence and functional relevance at the DNA and RNA level has been demonstrated in a number of viral, bacterial, and eukaryotic genomes, including humans. Here, we performed the first systematic search of G4-forming sequences in all archaeal genomes available in the NCBI database. In this article, we investigate the presence and locations of G-quadruplex forming sequences using the G4Hunter algorithm. G-quadruplex-prone sequences were identified in all archaeal species, with highly significant differences in frequency, from 0.037 to 15.31 potential quadruplex sequences per kb. While G4 forming sequences were extremely abundant in Hadesarchaea archeon (strikingly, more than 50% of the Hadesarchaea archaeon isolate WYZ-LMO6 genome is a potential part of a G4-motif), they were very rare in the Parvarchaeota phylum. The presence of G-quadruplex forming sequences does not follow a random distribution with an over-representation in non-coding RNA, suggesting possible roles for ncRNA regulation. These data illustrate the unique and non-random localization of G-quadruplexes in Archaea.
Assuntos
Archaea/genética , DNA/química , Quadruplex G , Genoma Arqueal/genética , RNA/química , Archaea/classificação , Archaea/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Dicroísmo Circular , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genômica/métodos , Conformação de Ácido Nucleico , Filogenia , RNA/genética , RNA/metabolismo , Especificidade da EspécieRESUMO
To extend the frontier of genome editing and enable editing of repetitive elements of mammalian genomes, we made use of a set of dead-Cas9 base editor (dBE) variants that allow editing at tens of thousands of loci per cell by overcoming the cell death associated with DNA double-strand breaks and single-strand breaks. We used a set of gRNAs targeting repetitive elements-ranging in target copy number from about 32 to 161 000 per cell. dBEs enabled survival after large-scale base editing, allowing targeted mutations at up to â¼13 200 and â¼12 200 loci in 293T and human induced pluripotent stem cells (hiPSCs), respectively, three orders of magnitude greater than previously recorded. These dBEs can overcome current on-target mutation and toxicity barriers that prevent cell survival after large-scale genome engineering.
Assuntos
Edição de Genes/métodos , Retroelementos , Proteínas Associadas a CRISPR , Sistemas CRISPR-Cas , Sobrevivência Celular , Endodesoxirribonucleases , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas , Mutação , RNARESUMO
Circular RNAs (circRNAs) differ structurally from other types of RNAs and are resistant against exoribonucleases. Although they have been detected in all domains of life, it remains unclear how circularization affects or changes functions of these ubiquitous nucleic acid circles. The biogenesis of circRNAs has been mostly described as a backsplicing event, but in archaea, where RNA splicing is a rare phenomenon, a second pathway for circRNA formation was described in the cases of rRNAs processing, tRNA intron excision, and Box C/D RNAs formation. At least in some archaeal species, circRNAs are formed by a ligation step catalyzed by an atypic homodimeric RNA ligase belonging to Rnl3 family. In this review, we describe archaeal circRNA transcriptomes obtained using high throughput sequencing technologies on Sulfolobus solfataricus, Pyrococcus abyssi and Nanoarchaeum equitans cells. We will discuss the distribution of circular RNAs among the different RNA categories and present the Rnl3 ligase family implicated in the circularization activity. Special focus is given for the description of phylogenetic distributions, protein structures, and substrate specificities of archaeal RNA ligases.
Assuntos
Nanoarchaeota , Pyrococcus abyssi , RNA Ligase (ATP) , RNA Arqueal , RNA Circular , Sulfolobus solfataricus , Nanoarchaeota/enzimologia , Nanoarchaeota/genética , Pyrococcus abyssi/enzimologia , Pyrococcus abyssi/genética , RNA Ligase (ATP)/classificação , RNA Ligase (ATP)/fisiologia , RNA Arqueal/classificação , RNA Arqueal/metabolismo , RNA Circular/classificação , RNA Circular/metabolismo , Análise de Sequência de RNA , Sulfolobus solfataricus/enzimologia , Sulfolobus solfataricus/genéticaRESUMO
Since the discovery of a flavin-dependent thymidylate synthase (ThyX or FDTS) that is absent in humans but crucial for DNA biosynthesis in a diverse group of pathogens, the enzyme has been pursued for the development of new antibacterial agents against Mycobacterium tuberculosis, the causative agent of the widespread infectious disease tuberculosis (TB). In response to a growing need for more effective anti-TB drugs, we have built upon our previous screening efforts and report herein an optimization campaign of a novel series of inhibitors with a unique inhibition profile. The inhibitors display competitive inhibition toward the methylene tetrahydrofolate cofactor of ThyX, enabling us to generate a model of the compounds bound to their target, thus offering insight into their structure-activity relationships.
Assuntos
Inibidores Enzimáticos , Mycobacterium tuberculosis/efeitos dos fármacos , Oxazinas , Timidilato Sintase/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Mycobacterium tuberculosis/enzimologia , Oxazinas/síntese química , Oxazinas/química , Oxazinas/farmacologia , Relação Estrutura-AtividadeRESUMO
INTRODUCTION: Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) remains the deadliest infectious disease in the world with one-third of the world's population thought to be infected. Over the years, TB mortality rate has been largely reduced; however, this progress has been threatened by the increasing appearance of multidrug-resistant Mtb. Considerable recent efforts have been undertaken to develop new generation antituberculosis drugs. Many of these attempts have relied on in silico approaches, which have emerged recently as powerful tools complementary to biochemical attempts. Areas covered: The authors review the status of pharmaceutical drug development against TB with a special emphasis on computational work. They focus on those studies that have been validated by in vitro and/or in vivo experiments, and thus, that can be considered as successful. The major goals of this review are to present target protein systems, to highlight how in silico efforts compliment experiments, and to aid future drug design endeavors. Expert opinion: Despite having access to all of the gene and protein sequences of Mtb, the search for new optimal treatments against this deadly pathogen are still ongoing. Together with the geometric growth of protein structural and sequence databases, computational methods have become a powerful technique accelerating the successful identification of new ligands.
Assuntos
Antituberculosos/administração & dosagem , Desenho de Fármacos , Tuberculose/tratamento farmacológico , Antituberculosos/farmacologia , Simulação por Computador , Desenvolvimento de Medicamentos/métodos , Humanos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose/microbiologiaRESUMO
Serine hydroxymethyltransferase (SHMT), encoded by the glyA gene, is a ubiquitous pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the formation of glycine from serine. The thereby generated 5,10-methylene tetrahydrofolate (MTHF) is a major source of cellular one-carbon units and a key intermediate in thymidylate biosynthesis. While in virtually all eukaryotic and many bacterial systems thymidylate synthase ThyA, SHMT and dihydrofolate reductase (DHFR) are part of the thymidylate/folate cycle, the situation is different in organisms using flavin-dependent thymidylate synthase ThyX. Here the distinct catalytic reaction directly produces tetrahydrofolate (THF) and consequently in most ThyX-containing organisms, DHFR is absent. While the resulting influence on the folate metabolism of ThyX-containing bacteria is not fully understood, the presence of ThyX may provide growth benefits under conditions where the level of reduced folate derivatives is compromised. Interestingly, the third key enzyme implicated in generation of MTHF, serine hydroxymethyltransferase (SHMT), has a universal phylogenetic distribution, but remains understudied in ThyX-containg bacteria. To obtain functional insight into these ThyX-dependent thymidylate/folate cycles, we characterized the predicted SHMT from the ThyX-containing bacterium Helicobacter pylori. Serine hydroxymethyltransferase activity was confirmed by functional genetic complementation of a glyA-inactivated E. coli strain. A H. pylori ΔglyA strain was obtained, but exhibited markedly slowed growth and had lost the virulence factor CagA. Biochemical and spectroscopic evidence indicated formation of a characteristic enzyme-PLP-glycine-folate complex and revealed unexpectedly weak binding affinity of PLP. The three-dimensional structure of the H. pylori SHMT apoprotein was determined at 2.8Ǻ resolution, suggesting a structural basis for the low affinity of the enzyme for its cofactor. Stabilization of the proposed inactive configuration using small molecules has potential to provide a specific way for inhibiting HpSHMT.
Assuntos
Proteínas de Bactérias , Glicina Hidroximetiltransferase , Helicobacter pylori , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Catálise , Escherichia coli/enzimologia , Escherichia coli/genética , Ácido Fólico/química , Ácido Fólico/genética , Ácido Fólico/metabolismo , Teste de Complementação Genética , Glicina/química , Glicina/genética , Glicina/metabolismo , Glicina Hidroximetiltransferase/química , Glicina Hidroximetiltransferase/genética , Glicina Hidroximetiltransferase/metabolismo , Helicobacter pylori/enzimologia , Helicobacter pylori/genética , Domínios ProteicosRESUMO
Using the haloarchaeon Haloferax volcanii as a model, we developed nascent DNA labeling and the functional GFP-labeled single-stranded binding protein RPA2 as novel tools to gain new insight into DNA replication and repair in live haloarchaeal cells. Our quantitative fluorescence microscopy data revealed that RPA2 forms distinct replication structures that dynamically responded to replication stress and DNA damaging agents. The number of the RPA2 foci per cell followed a probabilistic Poisson distribution, implying hitherto unnoticed stochastic cell-to-cell variation in haloarchaeal DNA replication and repair processes. The size range of haloarchaeal replication structures is very similar to those observed earlier in eukaryotic cells. The improved lateral resolution of 3D-SIM fluorescence microscopy allowed proposing that inhibition of DNA synthesis results in localized replication foci clustering and facilitated observation of RPA2 complexes brought about by chemical agents creating DNA double-strand breaks. Altogether our in vivo observations are compatible with earlier in vitro studies on archaeal single-stranded DNA binding proteins. Our work thus underlines the great potential of live cell imaging for unraveling the dynamic nature of transient molecular interactions that underpin fundamental molecular processes in the Third domain of life.
Assuntos
Reparo do DNA , Replicação do DNA/genética , DNA Arqueal/genética , Haloferax volcanii/genética , Microscopia de Fluorescência/métodos , Algoritmos , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Haloferax volcanii/citologia , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismoRESUMO
Comparative genome analyses have led to the discovery and characterization of novel flavin- and folate-dependent methyltransferases that mainly function in DNA precursor synthesis and post-transcriptional RNA modification by forming (ribo) thymidylate and its derivatives. Here we discuss the recent literature on the novel mechanistic features of these enzymes sometimes referred to as "uracil methyltransferases," albeit we prefer to refer to them as (ribo) thymidylate synthases. These enzyme families attest to the convergent evolution of nucleic acid methylation. Special focus is given to describing the unique characteristics of these flavin- and folate-dependent enzymes that have emerged as new models for studying the non-canonical roles of reduced flavin co-factors (FADH2) in relaying carbon atoms between enzyme substrates. This ancient enzymatic methylation mechanism with a very wide phylogenetic distribution may be more commonly used for biological methylation reactions than previously anticipated. This notion is exemplified by the recent discovery of additional substrates for these enzymes. Moreover, similar reaction mechanisms can be reversed by demethylases, which remove methyl groups e.g., from human histones. Future work is now required to address whether the use of different methyl donors facilitates the regulation of distinct methylation reactions in the cell. It will also be of great interest to address whether the low activity flavin-dependent thymidylate synthases ThyX represent ancestral enzymes that were eventually replaced by the more active thymidylate synthases of the ThyA family to facilitate the maintenance of larger genomes in fast-growing microbes. Moreover, we discuss the recent efforts from several laboratories to identify selective anti-microbial compounds that target flavin-dependent thymidylate synthase ThyX. Altogether we underline how the discovery of the alternative flavoproteins required for methylation of DNA and/or RNA nucleotides, in addition to providing novel targets for antibiotics, has provided new insight into microbial physiology and virulence.
RESUMO
The mismatch repair (MMR) system, exemplified by the MutS/MutL proteins, is widespread in Bacteria and Eukarya. However, molecular mechanisms how numerous archaea and bacteria lacking the mutS/mutL genes maintain high replication fidelity and genome stability have remained elusive. EndoMS is a recently discovered hyperthermophilic mismatch-specific endonuclease encoded by nucS in Thermococcales. We deleted the nucS from the actinobacterium Corynebacterium glutamicum and demonstrated a drastic increase of spontaneous transition mutations in the nucS deletion strain. The observed spectra of these mutations were consistent with the enzymatic properties of EndoMS in vitro. The robust mismatch-specific endonuclease activity was detected with the purified C. glutamicum EndoMS protein but only in the presence of the ß-clamp (DnaN). Our biochemical and genetic data suggest that the frequently occurring G/T mismatch is efficiently repaired by the bacterial EndoMS-ß-clamp complex formed via a carboxy-terminal sequence motif of EndoMS proteins. Our study thus has great implications for understanding how the activity of the novel MMR system is coordinated with the replisome and provides new mechanistic insight into genetic diversity and mutational patterns in industrially and clinically (e.g. Mycobacteria) important archaeal and bacterial phyla previously thought to be devoid of the MMR system.
Assuntos
Proteínas de Bactérias/metabolismo , Pareamento Incorreto de Bases , Corynebacterium glutamicum/enzimologia , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Actinobacteria/enzimologia , Proteínas de Bactérias/isolamento & purificação , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/isolamento & purificação , DNA/química , DNA/metabolismo , DNA Polimerase Dirigida por DNA/isolamento & purificação , MutaçãoRESUMO
In Algeria, many salt lakes are to be found spread from southern Tunisia up to the Atlas Mountains in northern Algeria. Oum Eraneb and Ain El beida sebkhas (salt lakes), are located in the Algerian Sahara. The aim of this study was to explore the diversity of the halobacteria in this type of habitats. The physicochemical properties of these shallow saline environments were examined and compared with other hypersaline and marine ecosystems. Both sites were relatively alkaline with a pH around 8.57- 8.74 and rich in salt at 13% and 16% (w/v) salinity for Oum Eraneb and Ain El beida, respectively, with dominant ions of sodium and chloride. The microbial approach revealed the presence of two halophilic archaea, strains JCM13561 and A33T in both explored sebkhas. Growth occurred between 10 and 25% (w/v) NaCl and the isolates grow optimally at 20% (w/v) NaCl. The pH range for growth was 6 to 9.5 with an optimum at pH 7.5 for the first strain and 7 to 9.5 with an optimum pH at 8.5-9 for the second strain. On the basis of 16S rRNA gene sequence analysis, strains JCM13561 and A33T were most closely related to Halorubrum litoreum and Natronorubrum bangense (99% and 96% similarity, respectively).
Assuntos
DNA Arqueal/genética , Halobacteriaceae/isolamento & purificação , Halorubrum/isolamento & purificação , Lagos/microbiologia , RNA Ribossômico 16S/genética , África do Norte , Argélia , Halobacteriaceae/classificação , Halobacteriaceae/efeitos dos fármacos , Halobacteriaceae/genética , Halorubrum/classificação , Halorubrum/efeitos dos fármacos , Halorubrum/genética , Concentração de Íons de Hidrogênio , Salinidade , Análise de Sequência de DNA , Cloreto de Sódio/farmacologiaRESUMO
Tyrosine (TyrOH) and tryptophan radicals play important roles as intermediates in biochemical charge-transfer reactions. Tryptophanyl radicals have been observed both in their protonated cation form and in their unprotonated neutral form, but to date, tyrosyl radicals have only been observed in their unprotonated form. With a genetically modified form of the flavoenzyme TrmFO as a suitable model system and using ultrafast fluorescence and absorption spectroscopy, we characterize its protonated precursor TyrOHâ¢+, and we show this species to have a distinct visible absorption band and a transition moment that we suggest to lie close to the phenol symmetry axis. TyrOHâ¢+ is formed in â¼1 ps by electron transfer to excited flavin and decays in â¼3 ps by charge recombination. These findings imply that TyrOH oxidation does not necessarily induce its concerted deprotonation. Our results will allow disentangling of photoproduct states in flavoproteins in often-encountered complex situations and more generally are important for understanding redox chains relying on tyrosyl intermediates.