RESUMO
Dihydrouridine (D), a prevalent and evolutionarily conserved base in the transcriptome, primarily resides in tRNAs and, to a lesser extent, in mRNAs. Notably, this modification is found at position 2449 in the Escherichia coli 23S rRNA, strategically positioned near the ribosome's peptidyl transferase site. Despite the prior identification, in E. coli genome, of three dihydrouridine synthases (DUS), a set of NADPH and FMN-dependent enzymes known for introducing D in tRNAs and mRNAs, characterization of the enzyme responsible for D2449 deposition has remained elusive. This study introduces a rapid method for detecting D in rRNA, involving reverse transcriptase-blockage at the rhodamine-labeled D2449 site, followed by PCR amplification (RhoRT-PCR). Through analysis of rRNA from diverse E. coli strains, harboring chromosomal or single-gene deletions, we pinpoint the yhiN gene as the ribosomal dihydrouridine synthase, now designated as RdsA. Biochemical characterizations uncovered RdsA as a unique class of flavoenzymes, dependent on FAD and NADH, with a complex structural topology. In vitro assays demonstrated that RdsA dihydrouridylates a short rRNA transcript mimicking the local structure of the peptidyl transferase site. This suggests an early introduction of this modification before ribosome assembly. Phylogenetic studies unveiled the widespread distribution of the yhiN gene in the bacterial kingdom, emphasizing the conservation of rRNA dihydrouridylation. In a broader context, these findings underscore nature's preference for utilizing reduced flavin in the reduction of uridines and their derivatives.
Assuntos
Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , RNA Ribossômico 23S/metabolismo , RNA Ribossômico 23S/genética , RNA Ribossômico 23S/química , Uridina/análogos & derivados , Uridina/metabolismo , Uridina/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , RNA Bacteriano/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/químicaRESUMO
Dihydrouridine (D) is a common modified base found predominantly in transfer RNA (tRNA). Despite its prevalence, the mechanisms underlying dihydrouridine biosynthesis, particularly in prokaryotes, have remained elusive. Here, we conducted a comprehensive investigation into D biosynthesis in Bacillus subtilis through a combination of genetic, biochemical, and epitranscriptomic approaches. Our findings reveal that B. subtilis relies on two FMN-dependent Dus-like flavoprotein homologs, namely DusB1 and DusB2, to introduce all D residues into its tRNAs. Notably, DusB1 exhibits multisite enzyme activity, enabling D formation at positions 17, 20, 20a and 47, while DusB2 specifically catalyzes D biosynthesis at positions 20 and 20a, showcasing a functional redundancy among modification enzymes. Extensive tRNA-wide D-mapping demonstrates that this functional redundancy impacts the majority of tRNAs, with DusB2 displaying a higher dihydrouridylation efficiency compared to DusB1. Interestingly, we found that BsDusB2 can function like a BsDusB1 when overexpressed in vivo and under increasing enzyme concentration in vitro. Furthermore, we establish the importance of the D modification for B. subtilis growth at suboptimal temperatures. Our study expands the understanding of D modifications in prokaryotes, highlighting the significance of functional redundancy in this process and its impact on bacterial growth and adaptation.
Assuntos
Bacillus subtilis , RNA de Transferência , Uridina , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , RNA Bacteriano/metabolismo , RNA Bacteriano/genética , RNA de Transferência/metabolismo , RNA de Transferência/genética , Uridina/metabolismo , Uridina/análogos & derivados , Expressão GênicaRESUMO
Dihydrouridine (D) is an abundant modified base found in the tRNAs of most living organisms and was recently detected in eukaryotic mRNAs. This base confers significant conformational plasticity to RNA molecules. The dihydrouridine biosynthetic reaction is catalyzed by a large family of flavoenzymes, the dihydrouridine synthases (Dus). So far, only bacterial Dus enzymes and their complexes with tRNAs have been structurally characterized. Understanding the structure-function relationships of eukaryotic Dus proteins has been hampered by the paucity of structural data. Here, we combined extensive phylogenetic analysis with high-precision 3D molecular modeling of more than 30 Dus2 enzymes selected along the tree of life to determine the evolutionary molecular basis of D biosynthesis by these enzymes. Dus2 is the eukaryotic enzyme responsible for the synthesis of D20 in tRNAs and is involved in some human cancers and in the detoxification of ß-amyloid peptides in Alzheimer's disease. In addition to the domains forming the canonical structure of all Dus, i.e., the catalytic TIM-barrel domain and the helical domain, both participating in RNA recognition in the bacterial Dus, a majority of Dus2 proteins harbor extensions at both ends. While these are mainly unstructured extensions on the N-terminal side, the C-terminal side extensions can adopt well-defined structures such as helices and beta-sheets or even form additional domains such as zinc finger domains. 3D models of Dus2/tRNA complexes were also generated. This study suggests that eukaryotic Dus2 proteins may have an advantage in tRNA recognition over their bacterial counterparts due to their modularity.
Assuntos
Oxirredutases , Uridina , Humanos , Bactérias/enzimologia , Bactérias/metabolismo , Eucariotos/enzimologia , Oxirredutases/química , Oxirredutases/classificação , Oxirredutases/genética , Filogenia , RNA de Transferência/metabolismo , Uridina/metabolismoRESUMO
Until recently, post-transcriptional modifications of RNA were largely restricted to noncoding RNA species. However, this belief seems to have quickly dissipated with the growing number of new modifications found in mRNA that were originally thought to be primarily tRNA-specific, such as dihydrouridine. Recently, transcriptomic profiling, metabolic labeling, and proteomics have identified unexpected dihydrouridylation of mRNAs, greatly expanding the catalog of novel mRNA modifications. These data also implicated dihydrouridylation in meiotic chromosome segregation, protein translation rates, and cell proliferation. Dihydrouridylation of tRNAs and mRNAs are introduced by flavin-dependent dihydrouridine synthases. In this review, we will briefly outline the current knowledge on the distribution of dihydrouridines in the transcriptome, their chemical labeling, and highlight structural and mechanistic aspects regarding the dihydrouridine synthases enzyme family. A special emphasis on important research directions to be addressed will also be discussed. This new entry of dihydrouridine into mRNA modifications has definitely added a new layer of information that controls protein synthesis.
Assuntos
RNA , Transcriptoma , Biossíntese de Proteínas , RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA de Transferência/químicaRESUMO
Dihydrouridine (D) is a tRNA-modified base conserved throughout all kingdoms of life and assuming an important structural role. The conserved dihydrouridine synthases (Dus) carries out D-synthesis. DusA, DusB and DusC are bacterial members, and their substrate specificity has been determined in Escherichia coli. DusA synthesizes D20/D20a while DusB and DusC are responsible for the synthesis of D17 and D16, respectively. Here, we characterize the function of the unique dus gene encoding a DusB detected in Mollicutes, which are bacteria that evolved from a common Firmicute ancestor via massive genome reduction. Using in vitro activity tests as well as in vivo E. coli complementation assays with the enzyme from Mycoplasma capricolum (DusBMCap), a model organism for the study of these parasitic bacteria, we show that, as expected for a DusB homolog, DusBMCap modifies U17 to D17 but also synthetizes D20/D20a combining therefore both E. coli DusA and DusB activities. Hence, this is the first case of a Dus enzyme able to modify up to three different sites as well as the first example of a tRNA-modifying enzyme that can modify bases present on the two opposite sides of an RNA-loop structure. Comparative analysis of the distribution of DusB homologs in Firmicutes revealed the existence of three DusB subgroups namely DusB1, DusB2 and DusB3. The first two subgroups were likely present in the Firmicute ancestor, and Mollicutes have retained DusB1 and lost DusB2. Altogether, our results suggest that the multisite specificity of the M. capricolum DusB enzyme could be an ancestral property.
Assuntos
Oxirredutases/metabolismo , RNA de Transferência/química , Tenericutes/genética , Uridina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Escherichia coli/genética , Evolução Molecular , Modelos Moleculares , Conformação de Ácido Nucleico , Oxirredutases/genética , RNA Bacteriano/química , Especificidade por Substrato , Tenericutes/metabolismoRESUMO
Extensive knowledge of both the nature and position of tRNA modifications in all cellular tRNAs has been limited to two bacteria, Escherichia coli and Mycoplasma capricolum. Bacillus subtilis sp subtilis strain 168 is the model Gram-positive bacteria and the list of the genes involved in tRNA modifications in this organism is far from complete. Mass spectrometry analysis of bulk tRNA extracted from B. subtilis, combined with next generation sequencing technologies and comparative genomic analyses, led to the identification of 41 tRNA modification genes with associated confidence scores. Many differences were found in this model Gram-positive bacteria when compared to E. coli. In general, B. subtilis tRNAs are less modified than those in E. coli, even if some modifications, such as m1A22 or ms2t6A, are only found in the model Gram-positive bacteria. Many examples of non-orthologous displacements and of variations in the most complex pathways are described. Paralog issues make uncertain direct annotation transfer from E. coli to B. subtilis based on homology only without further experimental validation. This difficulty was shown with the identification of the B. subtilis enzyme that introduces ψ at positions 31/32 of the tRNAs. This work presents the most up to date list of tRNA modification genes in B. subtilis, identifies the gaps in knowledge, and lays the foundation for further work to decipher the physiological role of tRNA modifications in this important model organism and other bacteria.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , RNA de Transferência/genética , Sequenciamento de Nucleotídeos em Larga Escala , Espectrometria de Massas , Conformação Molecular , RNA Bacteriano/genética , RNA de Transferência/química , Análise de Sequência de RNARESUMO
The C5-methylation of uracil to form 5-methyluracil (m5U) is a ubiquitous base modification of nucleic acids. Four enzyme families have converged to catalyze this methylation using different chemical solutions. Here, we investigate the evolution of 5-methyluracil synthase families in Mollicutes, a class of bacteria that has undergone extensive genome erosion. Many mollicutes have lost some of the m5U methyltransferases present in their common ancestor. Cases of duplication and subsequent shift of function are also described. For example, most members of the Spiroplasma subgroup use the ancestral tetrahydrofolate-dependent TrmFO enzyme to catalyze the formation of m5U54 in tRNA, while a TrmFO paralog (termed RlmFO) is responsible for m5U1939 formation in 23S rRNA. RlmFO has replaced the S-adenosyl-L-methionine (SAM)-enzyme RlmD that adds the same modification in the ancestor and which is still present in mollicutes from the Hominis subgroup. Another paralog of this family, the TrmFO-like protein, has a yet unidentified function that differs from the TrmFO and RlmFO homologs. Despite having evolved towards minimal genomes, the mollicutes possess a repertoire of m5U-modifying enzymes that is highly dynamic and has undergone horizontal transfer.
Assuntos
Evolução Molecular , Ácidos Nucleicos/metabolismo , Tenericutes/metabolismo , Uracila/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Sequência Conservada , Dinitrocresóis/metabolismo , Ácido Fólico/metabolismo , Metilação , Metiltransferases/metabolismo , Modelos Moleculares , RNA Ribossômico 23S/metabolismo , RNA de Transferência/metabolismo , Tenericutes/genéticaRESUMO
2'-O-Methylation (Nm) represents one of the most common RNA modifications. Nm affects RNA structure and function with crucial roles in various RNA-mediated processes ranging from RNA silencing, translation, self versus non-self recognition to viral defense mechanisms. Here, we identify two Nm methyltransferases (Nm-MTases) in Drosophila melanogaster (CG7009 and CG5220) as functional orthologs of yeast TRM7 and human FTSJ1. Genetic knockout studies together with MALDI-TOF mass spectrometry and RiboMethSeq mapping revealed that CG7009 is responsible for methylating the wobble position in tRNAPhe, tRNATrp and tRNALeu, while CG5220 methylates position C32 in the same tRNAs and also targets additional tRNAs. CG7009 or CG5220 mutant animals were viable and fertile but exhibited various phenotypes such as lifespan reduction, small RNA pathways dysfunction and increased sensitivity to RNA virus infections. Our results provide the first detailed characterization of two TRM7 family members in Drosophila and uncover a molecular link between enzymes catalyzing Nm at specific tRNAs and small RNA-induced gene silencing pathways.
Assuntos
Drosophila melanogaster/genética , Inativação Gênica , RNA de Transferência/genética , tRNA Metiltransferases/genética , Animais , Regulação da Expressão Gênica/genética , Humanos , Metilação , Metiltransferases/genética , Proteínas Nucleares/genética , Interferência de RNA , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Dihydrouridine (D) is an abundant modified base of tRNA found in the majority of living organisms. This base is synthesized via an NADPH-dependent reduction of specific uridines by the dihydrouridine synthases (Dus), a large family of flavoenzymes comprising eight subfamilies. Almost all of these enzymes function with only two conserved domains, an N-terminal catalytic domain (TBD) adopting a TIM barrel fold and a unique C-terminal helical domain (HD) devoted to tRNA recognition, except for the animal U20-specific Dus2 enzyme. Curiously, this enzyme is distinguished from paralogues and its fungi orthologues by the acquisition of an additional domain, a double stranded RNA binding domain (dsRBD), which serves as the main tRNA binding module. On the basis of a homology model of yeast Dus2 and the crystallographic structure of a human Dus2 variant (TBD + HD) lacking dsRBD, we herein show that the HD surface of the human enzyme is less electropositive than that of its yeast orthologue. This is partly due to two positively charged residues, K304 and K315, present in yeast and more broadly in fungi Dus2 that are replaced by E294 and Q305 in human and conserved among animals Dus2. By artificially reintroducing these positive charges in human Dus2 lacking dsRBD, we restored a functional tRNA binding in this enzyme variant. Altogether, these results suggest that the electrostatic potential changes of HD have likely played a key role in the emergence of a new tRNA binding mode among Dus2 enzymes.
Assuntos
Oxirredutases/metabolismo , RNA de Transferência/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Eletricidade Estática , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Evolução Molecular , Humanos , NADPH Oxidases/metabolismo , Oxirredutases/química , Oxirredutases/genética , Ligação Proteica , Conformação Proteica , RNA de Transferência/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por SubstratoRESUMO
Post-transcriptional base modifications are important to the maturation process of transfer RNAs (tRNAs). Certain modifications are abundant and present at several positions in tRNA as for example the dihydrouridine, a modified base found in the three domains of life. Even though the function of dihydrourine is not well understood, its high content in tRNAs from psychrophilic bacteria or cancer cells obviously emphasizes a central role in cell adaptation. The reduction of uridine to dihydrouridine is catalyzed by a large family of flavoenzymes named dihydrouridine synthases (Dus). Prokaryotes have three Dus (A, B and C) wherein DusB is considered as an ancestral protein from which the two others derived via gene duplications. Here, we unequivocally established the complete substrate specificities of the three Escherichia coli Dus and solved the crystal structure of DusB, enabling for the first time an exhaustive structural comparison between these bacterial flavoenzymes. Based on our results, we propose an evolutionary scenario explaining how substrate specificities has been diversified from a single structural fold.
Assuntos
Escherichia coli/química , Oxirredutases/química , RNA de Transferência/química , Uridina/análogos & derivados , Uridina/química , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli/enzimologia , Escherichia coli/genética , Evolução Molecular , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA de Transferência/genética , RNA de Transferência/metabolismo , Especificidade por Substrato , Termodinâmica , Uridina/metabolismoRESUMO
In tRNA, dihydrouridine is a conserved modified base generated by the post-transcriptional reduction of uridine. Formation of dihydrouridine 20, located in the D-loop, is catalyzed by dihydrouridine synthase 2 (Dus2). Human Dus2 (HsDus2) expression is upregulated in lung cancers, offering a growth advantage throughout its ability to interact with components of the translation apparatus and inhibit apoptosis. Here, we report the crystal structure of the individual domains of HsDus2 and their functional characterization. HsDus2 is organized into three major modules. The N-terminal catalytic domain contains the flavin cofactor involved in the reduction of uridine. The second module is the conserved α-helical domain known as the tRNA binding domain in HsDus2 homologues. It is connected via a flexible linker to an unusual extended version of a dsRNA binding domain (dsRBD). Enzymatic assays and yeast complementation showed that the catalytic domain binds selectively NADPH but cannot reduce uridine in the absence of the dsRBD. While in Dus enzymes from bacteria, plants and fungi, tRNA binding is essentially achieved by the α-helical domain, we showed that in HsDus2 this function is carried out by the dsRBD. This is the first reported case of a tRNA-modifying enzyme carrying a dsRBD used to bind tRNAs.
Assuntos
Oxirredutases/química , Processamento Pós-Transcricional do RNA , RNA de Transferência/metabolismo , Sítios de Ligação , Domínio Catalítico , Mononucleotídeo de Flavina/química , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , RNA de Transferência/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Methyltransferases that use S-adenosylmethionine (AdoMet) as a cofactor to catalyse 5-methyl uridine (m(5)U) formation in tRNAs and rRNAs are widespread in Bacteria and Eukaryota, and are also found in certain Archaea. These enzymes belong to the COG2265 cluster, and the Gram-negative bacterium Escherichia coli possesses three paralogues. These comprise the methyltransferases TrmA that targets U54 in tRNAs, RlmC that modifies U747 in 23S rRNA and RlmD that is specific for U1939 in 23S rRNA. The tRNAs and rRNAs of the Gram-positive bacterium Bacillus subtilis have the same three m(5)U modifications. However, as previously shown, the m(5)U54 modification in B. subtilis tRNAs is catalysed in a fundamentally different manner by the folate-dependent enzyme TrmFO, which is unrelated to the E. coli TrmA. Here, we show that methylation of U747 and U1939 in B. subtilis rRNA is catalysed by a single enzyme, YefA that is a COG2265 member. A recombinant version of YefA functions in an E. coli m(5)U-null mutant adding the same two rRNA methylations. The findings suggest that during evolution, COG2265 enzymes have undergone a series of changes in target specificity and that YefA is closer to an archetypical m(5)U methyltransferase. To reflect its dual specificity, YefA is renamed RlmCD.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Metiltransferases/metabolismo , RNA Ribossômico 23S/metabolismo , Uridina/análogos & derivados , Sequência de Aminoácidos , Proteínas de Bactérias/química , Biocatálise , Metiltransferases/química , Dados de Sequência Molecular , RNA Ribossômico 23S/química , Alinhamento de Sequência , Uridina/metabolismoRESUMO
The majority of human cells do not multiply continuously but are quiescent or slow-replicating and devote a large part of their energy to transcription. When DNA damage in the transcribed strand of an active gene is bypassed by a RNA polymerase, they can miscode at the damaged site and produce mutant transcripts. This process is known as transcriptional mutagenesis and, as discussed in this Perspective, could lead to the production of mutant proteins and might therefore be important in tumour development.
Assuntos
Mutagênese , Neoplasias/genética , Transcrição Gênica , Animais , Dano ao DNA , Replicação do DNA , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/metabolismo , Processos NeoplásicosRESUMO
Most of the somatic cells of adult metazoans, including mammals, do not undergo continuous cycles of replication. Instead, they are quiescent and devote most of their metabolic activity to gene expression. The mutagenic consequences of exposure to DNA-damaging agents are well documented, but less is known about the impact of DNA lesions on transcription. To investigate this impact, we developed a luciferase-based expression system. This system consists of two types of construct composed of a DNA template containing an 8-oxoguanine, paired either with a thymine or a cytosine, placed at defined positions along the transcribed strand of the reporter gene. Analyses of luciferase gene expression from the two types of construct showed that efficient but error-prone transcriptional bypass of 8-oxoguanine occurred in vivo, and that this lesion was not repaired by the transcription-coupled repair machinery in mammalian cells. The analysis of luciferase activity expressed from 8OG:T-containing constructs indicated that the magnitude of erroneous transcription events involving 8-oxoguanine depended on the sequence contexts surrounding the lesion. Additionally, sequencing of the transcript population expressed from these constructs showed that RNA polymerase II mostly inserted an adenine opposite to 8-oxoguanine. Analysis of luciferase expression from 8OG:C-containing constructs showed that the generated aberrant mRNAs led to the production of mutant proteins with the potential to induce a long-term phenotypical change. These findings reveal that erroneous transcription over DNA lesions may induce phenotypical changes with the potential to alter the fate of non-replicating cells.
Assuntos
Dano ao DNA , Mutagênese , Transcrição Gênica , Animais , Linhagem Celular , Reparo do DNA , Guanina/análogos & derivados , Humanos , Camundongos , MutagênicosRESUMO
Cells exposed to DNA-damaging agents in their natural environment do not undergo continuous cycles of replication but are more frequently engaged in gene transcription. Despite the relatively high efficiency of the different DNA repair pathways, some lesions remain in DNA. During transcription, RNA polymerase can bypass DNA damage on the transcribed strand of an active gene. This bypass can be at the origin of the production of "mutated" mRNA because of the transcriptional miscoding (transcriptional mutagenesis) due to the altered pairing specificities of the lesion. In vivo consequences of transcriptional mutagenesis on normal cell physiology have not well been documented because of the lack of a robust system allowing for its study. We describe here a procedure that we developed using a plasmid-based luciferase reporter assay to analyze the transcriptional mutagenesis events induced by different types of DNA lesions. Introduction of the DNA lesion to be studied at a specific site on the plasmid is based on the synthesis of a complementary strand of a circular, single-stranded DNA (ssDNA) from a DNA lesion-containing oligonucleotide. Once obtained, this construct can be transformed into different Escherichia coli strains that can express the luciferase gene under nongrowth conditions. Quantification of luciferase activity and sequencing of luciferase cDNAs allow for the characterization of transcriptional mutagenesis both quantitatively and qualitatively.
Assuntos
DNA Circular/genética , Mutagênese , Transcrição Gênica , Sequência de Bases , Primers do DNA , Replicação do DNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Most of nucleic acids damaging agents are not only restricted to DNA but equally affect DNA and RNA molecules. Considering that RNA damage could be very toxic for the cell, a property used by some cancer treatments, it would not be unexpected to find out that several proteins may be involved in RNA damage avoidance mechanisms helping cells to counteract such cytotoxic effects. Up to now, only one specific repair mechanism allowing cells to deal with toxic effects of methylated RNA have been described. However, there are in the literature several data suggesting that this study may only be the tip of the iceberg and that cells might be able to counteract the deleterious effects of a large variety of RNA damage. In this review, we will discuss the different proteins that may be involved in the mechanism of RNA damage avoidance and their potential role in human diseases.
Assuntos
RNA/genética , Alquilação , Humanos , Estresse Oxidativo , Raios UltravioletaRESUMO
Cells of all living organisms are continuously exposed to physical and chemical agents that damage DNA and alter the integrity of their genomes. Despite the relatively high efficiency of the different repair pathways, some lesions remain in DNA when it is replicated or transcribed. Lesion bypass by DNA and RNA polymerases has been the subject of numerous investigations. However, knowledge of the in vivo mechanism of transcription lesion bypass is very limited because no robust methodology is available. Here we describe a protocol based on the synthesis of a complementary strand of a circular, single-stranded DNA molecule, which allows for the production of large amounts of double-stranded DNA containing a lesion at a specific position in a transcribed sequence. Such constructs can subsequently be used for lesion bypass studies in vivo by RNA polymerase and to ascertain how these events can be affected by the genetic background of the cells.
Assuntos
Pareamento Incorreto de Bases/genética , Dano ao DNA , DNA/genética , Engenharia Genética/métodos , Vetores Genéticos/genética , Mutagênese Sítio-Dirigida/genética , Mutação , Reprodutibilidade dos TestesRESUMO
Cells exposed to DNA damaging agents in their natural environment do not undergo continuous cycles of replication but are more frequently engaged in gene transcription. Luciferase gene expression analysis with DNA templates containing uracil or 8-oxoguanine, placed at a defined position, indicated that in nondividing Escherichia coli cells, efficient mutagenic lesion bypass does occur in vivo during transcription. Sequence analyses of the transcript population revealed that RNA polymerase inserts adenine opposite to uracil, and adenine or cytosine opposite to 8-oxoguanine. Surprisingly, deletions were also detected for 8-oxoguanine-containing templates, indicating RNA polymerase slippage over this lesion. Genetic analyses showed that, in E. coli, 8-oxoguanine is subject to transcription-coupled repair. Consequently, DNA damages alter transcription fidelity in vivo, which may lead to the production of mutant proteins that have the potential to change the phenotype of nondividing cells.