RESUMO
Chromosome duplication initiates via the assembly of replication forks at defined origins. Forks proceed in opposite directions until they fuse with a converging fork. Recent work highlights that fork fusions are highly choreographed both in pro- and eukaryotic cells. The circular Escherichia coli chromosome is replicated from a single origin (oriC), and a single fork fusion takes place in a specialised termination area opposite oriC that establishes a fork trap mediated by Tus protein bound at ter sequences that allows forks to enter but not leave. Here we further define the molecular details of fork fusions and the role of RecG helicase in replication termination. Our data support the idea that fork fusions have the potential to trigger local re-replication of the already replicated DNA. In ΔrecG cells this potential is realised in a substantial fraction of cells and is dramatically elevated when one fork is trapped for some time before the converging fork arrives. They also support the idea that the termination area evolved to contain such over-replication and we propose that the stable arrest of replication forks at ter/Tus complexes is an important feature that limits the likelihood of problems arising as replication terminates.
Assuntos
Cromossomos Bacterianos/genética , Replicação do DNA/genética , DNA Bacteriano/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Genéticos , Mutação , Conformação de Ácido Nucleico , Origem de Replicação/genéticaRESUMO
Chromosome duplication normally initiates through the assembly of replication fork complexes at defined origins. DNA synthesis by any one fork is thought to cease when it meets another travelling in the opposite direction, at which stage the replication machinery may simply dissociate before the nascent strands are finally ligated. But what actually happens is not clear. Here we present evidence consistent with the idea that every fork collision has the potential to threaten genomic integrity. In Escherichia coli this threat is kept at bay by RecG DNA translocase and by single-strand DNA exonucleases. Without RecG, replication initiates where forks meet through a replisome assembly mechanism normally associated with fork repair, replication restart and recombination, establishing new forks with the potential to sustain cell growth and division without an active origin. This potential is realized when roadblocks to fork progression are reduced or eliminated. It relies on the chromosome being circular, reinforcing the idea that replication initiation is triggered repeatedly by fork collision. The results reported raise the question of whether replication fork collisions have pathogenic potential for organisms that exploit several origins to replicate each chromosome.
Assuntos
Cromossomos Bacterianos/metabolismo , Replicação do DNA , Escherichia coli/genética , Origem de Replicação , Proteínas de Bactérias/metabolismo , Bacteriófagos/genética , Bacteriófagos/fisiologia , Cromossomos Bacterianos/genética , DNA Helicases/metabolismo , DNA Bacteriano , DNA Circular , DNA de Cadeia Simples , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/enzimologia , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/virologia , Proteínas de Escherichia coli/metabolismo , Exodesoxirribonuclease V/metabolismo , Exonucleases/metabolismo , Marcadores Genéticos/genética , Instabilidade GenômicaRESUMO
Conflicts between replication and transcription challenge chromosome duplication. Escherichia coli replisome movement along transcribed DNA is promoted by Rep and UvrD accessory helicases with Δrep ΔuvrD cells being inviable under rapid growth conditions. We have discovered that mutations in a tRNA gene, aspT, in an aminoacyl tRNA synthetase, AspRS, and in a translation factor needed for efficient proline-proline bond formation, EF-P, suppress Δrep ΔuvrD lethality. Thus replication-transcription conflicts can be alleviated by the partial sacrifice of a mechanism that reduces replicative barriers, namely translating ribosomes that reduce RNA polymerase backtracking. Suppression depends on RelA-directed synthesis of (p)ppGpp, a signalling molecule that reduces replication-transcription conflicts, with RelA activation requiring ribosomal pausing. Levels of (p)ppGpp in these suppressors also correlate inversely with the need for Rho activity, an RNA translocase that can bind to emerging transcripts and displace transcription complexes. These data illustrate the fine balance between different mechanisms in facilitating gene expression and genome duplication and demonstrate that accessory helicases are a major determinant of this balance. This balance is also critical for other aspects of bacterial survival: the mutations identified here increase persistence indicating that similar mutations could arise in naturally occurring bacterial populations facing antibiotic challenge.
Assuntos
Replicação do DNA , Escherichia coli/genética , Genoma Bacteriano , Elongação Traducional da Cadeia Peptídica , DNA Helicases/genética , Escherichia coli/enzimologia , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mutação , RNA de Transferência de Ácido Aspártico/genética , Supressão Genética , Aminoacilação de RNA de TransferênciaRESUMO
Nucleoprotein complexes present challenges to genome stability by acting as potent blocks to replication. One attractive model of how such conflicts are resolved is direct targeting of blocked forks by helicases with the ability to displace the blocking protein-DNA complex. We show that Rep and UvrD each promote movement of E. coli replisomes blocked by nucleoprotein complexes in vitro, that such an activity is required to clear protein blocks (primarily transcription complexes) in vivo, and that a polarity of translocation opposite that of the replicative helicase is critical for this activity. However, these two helicases are not equivalent. Rep but not UvrD interacts physically and functionally with the replicative helicase. In contrast, UvrD likely provides a general means of protein-DNA complex turnover during replication, repair, and recombination. Rep and UvrD therefore provide two contrasting solutions as to how organisms may promote replication of protein-bound DNA.
Assuntos
DNA Helicases/metabolismo , DNA Bacteriano/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas Motores Moleculares/metabolismo , Complexos Multienzimáticos/metabolismo , Meios de Cultura , Replicação do DNA , DnaB Helicases/metabolismo , Escherichia coli/citologia , Escherichia coli/enzimologia , Escherichia coli/crescimento & desenvolvimento , Teste de Complementação Genética , Mutação/genética , Nucleoproteínas/metabolismo , Ligação Proteica , Supressão Genética , Transcrição GênicaRESUMO
The RecG protein of Escherichia coli is a double-stranded DNA translocase that unwinds a variety of branched substrates in vitro. Although initially associated with homologous recombination and DNA repair, studies of cells lacking RecG over the past 25 years have led to the suggestion that the protein might be multi-functional and associated with a number of additional cellular processes, including initiation of origin-independent DNA replication, the rescue of stalled or damaged replication forks, replication restart, stationary phase or stress-induced 'adaptive' mutations and most recently, naïve adaptation in CRISPR-Cas immunity. Here we discuss the possibility that many of the phenotypes of recG mutant cells that have led to this conclusion may stem from a single defect, namely the failure to prevent re-replication of the chromosome. We also present data indicating that this failure does indeed contribute substantially to the much-reduced recovery of recombinants in conjugational crosses with strains lacking both RecG and the RuvABC Holliday junction resolvase.
Assuntos
DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Cromossomos Bacterianos , Dano ao DNA , Replicação do DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Genótipo , Recombinação Homóloga , MutaçãoRESUMO
RecG is a DNA translocase encoded by most species of bacteria. The Escherichia coli protein targets branched DNA substrates and drives the unwinding and rewinding of DNA strands. Its ability to remodel replication forks and to genetically interact with PriA protein have led to the idea that it plays an important role in securing faithful genome duplication. Here we report that RecG co-localises with sites of DNA replication and identify conserved arginine and tryptophan residues near its C-terminus that are needed for this localisation. We establish that the extreme C-terminus, which is not resolved in the crystal structure, is vital for DNA unwinding but not for DNA binding. Substituting an alanine for a highly conserved tyrosine near the very end results in a substantial reduction in the ability to unwind replication fork and Holliday junction structures but has no effect on substrate affinity. Deleting or substituting the terminal alanine causes an even greater reduction in unwinding activity, which is somewhat surprising as this residue is not uniformly present in closely related RecG proteins. More significantly, the extreme C-terminal mutations have little effect on localisation. Mutations that do prevent localisation result in only a slight reduction in the capacity for DNA repair.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas da Membrana Bacteriana Externa/metabolismo , Dano ao DNA , Replicação do DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/química , Genes Bacterianos , Genes Letais , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Transporte ProteicoRESUMO
Replication fork pausing drives genome instability, because any loss of paused replisome activity creates a requirement for reloading of the replication machinery, a potentially mutagenic process. Despite this importance, the relative contributions to fork pausing of different replicative barriers remain unknown. We show here that Deinococcus radiodurans RecD2 helicase inactivates Escherichia coli replisomes that are paused but still functional in vitro, preventing continued fork movement upon barrier removal or bypass, but does not inactivate elongating forks. Using RecD2 to probe replisome pausing in vivo, we demonstrate that most pausing events do not lead to replisome inactivation, that transcription complexes are the primary sources of this pausing, and that an accessory replicative helicase is critical for minimizing the frequency and/or duration of replisome pauses. These findings reveal the hidden potential for replisome inactivation, and hence genome instability, inside cells. They also demonstrate that efficient chromosome duplication requires mechanisms that aid resumption of replication by paused replisomes, especially those halted by protein-DNA barriers such as transcription complexes.
Assuntos
Replicação do DNA , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Morte Celular , DNA Helicases/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Complexos Multienzimáticos/metabolismo , Nucleoproteínas/metabolismo , Ligação Proteica , Transcrição GênicaRESUMO
RecG is a DNA translocase that helps to maintain genomic integrity. Initial studies suggested a role in promoting recombination, a possibility consistent with synergism between recG and ruv null alleles and reinforced when the protein was shown to unwind Holliday junctions. In this article we describe novel suppressors of recG and show that the pathology seen without RecG is suppressed on reducing or eliminating PriB, a component of the PriA system for replisome assembly and replication restart. Suppression is conditional, depending on additional mutations that modify ribosomal subunit S6 or one of three subunits of RNA polymerase. The latter suppress phenotypes associated with deletion of priB, enabling the deletion to suppress recG. They include alleles likely to disrupt interactions with transcription anti-terminator, NusA. Deleting priB has a different effect in ruv strains. It provokes abortive recombination and compromises DNA repair in a manner consistent with PriB being required to limit exposure of recombinogenic ssDNA. This synergism is reduced by the RNA polymerase mutations identified. Taken together, the results reveal that RecG curbs a potentially negative effect of proteins that direct replication fork assembly at sites removed from the normal origin, a facility needed to resolve conflicts between replication and transcription.
Assuntos
Proteínas de Bactérias/genética , Dano ao DNA , DNA Helicases/genética , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Endodesoxirribonucleases/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Proteínas de Bactérias/metabolismo , DNA Helicases/metabolismo , Reparo do DNA , Replicação do DNA , Proteínas de Ligação a DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Mutação , Recombinação Genética , Ribossomos/genética , Ribossomos/metabolismoRESUMO
Genome duplication requires accessory helicases to displace proteins ahead of advancing replication forks. Escherichia coli contains three helicases, Rep, UvrD and DinG, that might promote replication of protein-bound DNA. One of these helicases, Rep, also interacts with the replicative helicase DnaB. We demonstrate that Rep is the only putative accessory helicase whose absence results in an increased chromosome duplication time. We show also that the interaction between Rep and DnaB is required for Rep to maintain rapid genome duplication. Furthermore, this Rep-DnaB interaction is critical in minimizing the need for both recombinational processing of blocked replication forks and replisome reassembly, indicating that colocalization of Rep and DnaB minimizes stalling and subsequent inactivation of replication forks. These data indicate that E. coli contains only one helicase that acts as an accessory motor at the fork in wild-type cells, that such an activity is critical for the maintenance of rapid genome duplication and that colocalization with the replisome is crucial for this function. Given that the only other characterized accessory motor, Saccharomyces cerevisiae Rrm3p, associates physically with the replisome, our demonstration of the functional importance of such an association indicates that colocalization may be a conserved feature of accessory replicative motors.
Assuntos
DNA Helicases/metabolismo , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DnaB Helicases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Complexos Multienzimáticos/metabolismo , DNA Helicases/análise , DnaB Helicases/análise , Escherichia coli/enzimologia , Proteínas de Escherichia coli/análise , Genoma Bacteriano , Transcrição GênicaRESUMO
BACKGROUND: Manipulations of the DNA double helix during replication, transcription and other nucleic acid processing cause a change of DNA topology, which results in torsional stress. This stress is relaxed by DNA topoisomerases, a class of enzymes present in all domains of life. Negatively supercoiled DNA is relaxed by type IA topoisomerases that are widespread in bacteria, archaea and eukaryotes. In Escherichia coli there is conflicting data about viability of ΔtopA cells lacking topoisomerase I. RESULTS: In this study we sought to clarify whether E. coli cells lacking topoisomerase I are viable by using a plasmid-based lethality assay that allowed us to investigate the phenotype of ΔtopA cells without the presence of any compensatory mutations. Our results show that cells lacking topoisomerase I show an extreme growth defect and cannot be cultured without the accumulation of compensatory mutations. This growth defect can be partially suppressed by overexpression of topoisomerase III, the other type IA topoisomerase in E. coli, suggesting that the accumulation of torsional stress is, at least partially, responsible for the lethality of ΔtopA cells. The absence of RNase HI strongly exacerbates the phenotype of cells lacking topoisomerase I, which supports the idea that the processing of RNA:DNA hybrids is vitally important in ΔtopA cells. However, we did not observe suppression of the ΔtopA phenotype by increasing the level of R-loop processing enzymes, such as RNase HI or RecG. CONCLUSIONS: Our data show unambiguously that E. coli cells are not viable in the absence of DNA topoisomerase I without the presence of compensatory mutations. Furthermore, our data suggest that the accumulation of R-loops is not the primary reason for the severe growth defect of cells lacking topoisomerase I, which is in contrast to the current literature. Potential reasons for this discrepancy are discussed.
Assuntos
DNA Topoisomerases Tipo I/deficiência , Escherichia coli/enzimologia , Escherichia coli/fisiologia , Deleção de Genes , Viabilidade Microbiana , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Expressão Gênica , Ribonuclease H/genética , Ribonuclease H/metabolismo , Supressão GenéticaRESUMO
The DNA-binding protein RdgC has been identified as an inhibitor of RecA-mediated homologous recombination in Escherichia coli. In Neisseria species, RdgC also has a role in virulence-associated antigenic variation. We have previously solved the crystal structure of the E. coli RdgC protein and shown it to form a toroidal dimer. In this study, we have conducted a mutational analysis of residues proposed to mediate interactions at the dimer interfaces. We demonstrate that destabilizing either interface has a serious effect on in vivo function, even though a stable complex with circular DNA was still observed. We conclude that tight binding is required for inhibition of RecA activity. We also investigated the role of the RdgC finger domain, and demonstrate that it plays a crucial role in the binding of circular DNA. Together, these data allow us to propose a model for how RdgC loads onto DNA. We discuss how RdgC might inhibit RecA-mediated strand exchange, and how RdgC might be displaced by other DNA metabolism enzymes such as polymerases and helicases.
Assuntos
DNA Circular/metabolismo , Proteínas de Escherichia coli/química , Sítios de Ligação , DNA Helicases/genética , Dimerização , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Deleção de Genes , Modelos Moleculares , Mutação , Plasmídeos/metabolismo , Estrutura Terciária de ProteínaRESUMO
Duplication and transmission of chromosomes require precise control of chromosome replication and segregation. Here we present evidence that RecG is a major factor influencing these processes in bacteria. We show that the extensive DnaA-independent stable DNA replication observed without RecG can lead to replication of any area of the chromosome. This replication is further elevated following irradiation with UV light and appears to be perpetuated by secondary events that continue long after the elimination of UV lesions. The resulting pathological cascade is associated with an increased number of replication forks traversing the chromosome, sometimes with extensive regional amplification of the chromosome, and with the accumulation of highly branched DNA intermediates containing few Holliday junctions. We propose that the cascade is triggered by replication fork collisions that generate 3' single-strand DNA flaps, providing sites for PriA to initiate re-replication of the DNA and thus to generate linear duplexes that provoke recombination, allowing priming of even further replication. Our results shed light on why termination of replication in bacteria is normally limited to a single encounter of two forks and carefully orchestrated within a restricted area, and explain how a system of multiple forks and random termination can operate in eukaryotes.
Assuntos
Segregação de Cromossomos , Cromossomos Bacterianos/metabolismo , Replicação do DNA , Proteínas de Escherichia coli/fisiologia , Escherichia coli/enzimologia , Escherichia coli/fisiologia , DNA Cruciforme/metabolismo , Escherichia coli/efeitos da radiação , Proteínas de Escherichia coli/genética , Raios UltravioletaRESUMO
Little is known about what happens when forks meet to complete DNA replication in any organism. In this study we present data suggesting that the collision of replication forks is a potential threat to genomic stability. We demonstrate that Escherichia coli cells lacking RecG helicase suffer major defects in chromosome replication following UV irradiation, and that this is associated with high levels of DNA synthesis initiated independently of the initiator protein DnaA. This UV-induced stable DNA replication is dependent on PriA helicase and continues long after UV-induced lesions have been excised. We suggest UV irradiation triggers the assembly of new replication forks, leading to multiple fork collisions outside the terminus area. Such collisions may generate branched DNAs that serve to establish further new forks, resulting in uncontrolled DNA amplification. We propose that RecG reduces the likelihood of this pathological cascade being set in motion by reducing initiation of replication at D- and R-loops, and other structures generated as a result of fork collisions. Our results shed light on why replication initiation in bacteria is limited to a single origin and why termination is carefully orchestrated to a single event within a restricted area each cell cycle.
Assuntos
DNA Helicases/metabolismo , Replicação do DNA , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Divisão Celular , Cromossomos Bacterianos/genética , Dano ao DNA , DNA Helicases/genética , Replicação do DNA/efeitos da radiação , DNA Bacteriano/biossíntese , Escherichia coli/citologia , Escherichia coli/metabolismo , Escherichia coli/efeitos da radiação , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Raios UltravioletaRESUMO
In dividing cells, the stalling of replication fork complexes by impediments to DNA unwinding or by template imperfections that block synthesis by the polymerase subunits is a serious threat to genomic integrity and cell viability. What happens to stalled forks depends on the nature of the offending obstacle. In UV-irradiated Escherichia coli cells DNA synthesis is delayed for a considerable period, during which forks undergo extensive processing before replication can resume. Thus, restart depends on factors needed to load the replicative helicase, indicating that the replisome may have dissociated. It also requires the RecFOR proteins, which are known to load RecA recombinase on single-stranded DNA, implying that template strands are exposed. To gain a further understanding of how UV irradiation affects replication and how replication resumes after a block, we used fluorescence microscopy and BrdU or radioisotope labelling to examine chromosome replication and cell cycle progression. Our studies confirm that RecFOR promote efficient reactivation of stalled forks and demonstrate that they are also needed for productive replication initiated at the origin, or triggered elsewhere by damage to the DNA. Although delayed, all modes of replication do recover in the absence of these proteins, but nascent DNA strands are degraded more extensively by RecJ exonuclease. However, these strands are also degraded in the presence of RecFOR when restart is blocked by other means, indicating that RecA loading is not sufficient to stabilise and protect the fork. This is consistent with the idea that RecA actively promotes restart. Thus, in contrast to eukaryotic cells, there may be no factor in bacterial cells acting specifically to stabilise stalled forks. Instead, nascent strands may be protected by the simple expedient of promoting restart. We also report that the efficiency of fork reactivation is not affected in polB mutants.
Assuntos
DNA Polimerase II/fisiologia , Reparo do DNA , Replicação do DNA , Escherichia coli/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Escherichia coli/efeitos da radiação , Proteínas de Escherichia coli/genética , Mutação , Recombinases Rec A/genética , Origem de Replicação , Raios UltravioletaRESUMO
DNA double-strand breaks threaten the stability of the genome, and yet are induced deliberately during meiosis in order to provoke homologous recombination and generate the crossovers needed to promote faithful chromosome transmission. Crossovers are secured via biased resolution of the double Holliday junction intermediates formed when both ends of the broken chromosome engage an intact homologue. To investigate whether the enzymes catalysing branch migration and resolution of Holliday junctions are directed to favour production of either crossover or noncrossover products, we engineered a genetic system based on DNA breakage induced by the I-SceI endonuclease to detect analogous exchanges in Escherichia coli where the enzymology of recombination is more fully understood. Analysis of the recombinants generated revealed approximately equal numbers of crossover and noncrossover products, regardless of whether repair is mediated via RecG, RuvABC, or the RusA resolvase. We conclude that there little or no control of crossing over at the level of Holliday junction resolution. Thus, if similar resolvases function during meiosis, additional factors must act to bias recombination in favour of crossover progeny.
Assuntos
Troca Genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Resolvases de Junção Holliday/fisiologia , Proteínas de Bactérias/fisiologia , DNA Helicases , Endodesoxirribonucleases/fisiologia , Proteínas de Escherichia coli/fisiologia , Exodesoxirribonucleases/fisiologiaRESUMO
The premature aging and cancer-prone disease Werner syndrome stems from loss of WRN protein function. WRN deficiency causes replication abnormalities, sensitivity to certain genotoxic agents, genomic instability and early replicative senescence in primary fibroblasts. As a RecQ helicase family member, WRN is a DNA-dependent ATPase and unwinding enzyme, but also possesses strand annealing and exonuclease activities. RecQ helicases are postulated to participate in pathways responding to replication blockage, pathways possibly initiated by fork regression. In this study, a series of model replication fork substrates were used to examine the fork regression capability of WRN. Our results demonstrate that WRN catalyzes fork regression and Holliday junction formation. This process is an ATP-dependent reaction that is particularly efficient on forks containing single-stranded gaps of at least 11-13 nt on the leading arm at the fork junction. Importantly, WRN exonuclease activity, by digesting the leading daughter strand, enhances regression of forks with smaller gaps on the leading arm, thus creating an optimal structure for regression. Our results suggest that the multiple activities of WRN cooperate to promote replication fork regression. These findings, along with the established cellular consequences of WRN deficiency, strongly support a role for WRN in regression of blocked replication forks.
Assuntos
Replicação do DNA , DNA Cruciforme/metabolismo , Exodesoxirribonucleases/metabolismo , RecQ Helicases/metabolismo , Adenosina Trifosfatases/metabolismo , DNA/química , DNA/metabolismo , DNA Helicases/metabolismo , DNA Cruciforme/química , Exodesoxirribonucleases/fisiologia , Humanos , RecQ Helicases/fisiologia , Helicase da Síndrome de WernerRESUMO
DNA replication and transcription are essential processes for cell growth and division. The polymerase complexes charged with these tasks share the same template and so there is a potential for conflict between the two. Studies both in vivo and in vitro indicate that RNA polymerase complexes can indeed arrest the progress of replication forks and that cell viability is endangered as a result. Stable ternary complexes formed at lesions in the template DNA or as a result of backtracking may be especially troublesome. Numerous processes have evolved to reduce conflict between replication and transcription and to rescue any replication forks that suffer damage when conflict does occur. In bacteria, these systems are so highly tuned and co-ordinated that cells normally suffer few problems even under conditions allowing rapid growth and therefore demanding both efficient chromosome duplication and high levels of gene expression. The threat to genome transmission and cell viability becomes apparent when this interplay is disrupted.
Assuntos
Reparo do DNA , Replicação do DNA , RNA Polimerases Dirigidas por DNA , Transcrição Gênica , Animais , HumanosRESUMO
We have determined the structure of a catalytically inactive D70N variant of the Escherichia coli RusA resolvase bound to a duplex DNA substrate that reveals critical protein-DNA interactions and permits a much clearer understanding of the interaction of the enzyme with a Holliday junction (HJ). The RusA enzyme cleaves HJs, the fourway DNA branchpoints formed by homologous recombination, by introducing symmetrical cuts in the phosphodiester backbone in a Mg2+ dependent reaction. Although, RusA shows a high level of selectivity for DNA junctions, preferring to bind fourway junctions over other substrates in vitro, it has also been shown to have appreciable affinity for duplex DNA. However, RusA does not show DNA cleavage activity with duplex substrates. Our structure suggests the possible basis for structural selectivity as well as sources of the sequence specificity observed for DNA cleavage by RusA.
Assuntos
DNA Cruciforme/química , Proteínas de Escherichia coli/química , Resolvases de Junção Holliday/química , Modelos Moleculares , Substituição de Aminoácidos , Sítios de Ligação , DNA/química , DNA Cruciforme/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Resolvases de Junção Holliday/genética , Resolvases de Junção Holliday/metabolismo , Especificidade por SubstratoRESUMO
Chromosomal duplication faces many blocks to replication fork progression that could destabilize the genome and prove fatal if not overcome. Overcoming such blocks requires interplay between DNA replication, recombination and repair. The RecG protein of Escherichia coli promotes rescue of damaged forks by catalysing their unwinding and conversion to Holliday junctions. Subsequent processing of this structure allows repair or bypass of the fork block, enabling replication to resume without recourse to potentially mutagenic translesion synthesis or recombination. Such direct rescue of stalled forks might help safeguard genome integrity in all organisms.
Assuntos
Dano ao DNA , DNA Helicases , Reparo do DNA , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Genoma Bacteriano , Proteína de Replicação ARESUMO
Maintaining the integrity of the genome is essential for the survival of all organisms. RecG helicase plays an important part in this process in Escherichia coli, promoting recombination and DNA repair, and providing ways to rescue stalled replication forks by way of a Holliday junction intermediate. We purified RecG proteins from three other species: two Gram-positive mesophiles, Bacillus subtilis and Streptococcus pneumoniae, and one extreme thermophile, Aquifex aeolicus. All three proteins bind and unwind replication fork and Holliday junction DNA molecules with efficiencies similar to the E. coli protein. Proteins from the Gram-positive species promote DNA repair in E. coli, indicating either that RecG acts alone or that any necessary protein-protein interactions are conserved. The S. pneumoniae RecG reduces plasmid copy number when expressed in E. coli, indicating that like the E. coli protein it unwinds plasmid R loop structures used to prime replication. This effect is not seen with B. subtilis RecG; the protein either lacks R loop unwinding activity or is compromised by having insufficient ATP. The A. aeolicus protein unwinds DNA well at 60 degrees C but is less efficient at 37 degrees C, explaining its inability to function in E. coli at this temperature. The N-terminal extension present in this protein was investigated and found to be dispensable for activity and thermo-stability. The results presented suggest that the role of RecG in DNA replication and repair is likely to be conserved throughout all bacteria, which underlines the importance of this protein in genome duplication and cell survival.