RESUMO
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
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
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
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
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.
Assuntos
Bactérias/genética , DNA Helicases/fisiologia , Reparo do DNA , Replicação do DNA/fisiologia , Proteínas de Escherichia coli/fisiologia , Expressão Gênica , Bactérias/efeitos da radiação , Pareamento de Bases , Sequência de Bases , Biologia Computacional , DNA Helicases/genética , Ensaio de Desvio de Mobilidade Eletroforética , Proteínas de Escherichia coli/genética , Homologia de Sequência , Especificidade da Espécie , Temperatura , Raios UltravioletaRESUMO
Replication of the Escherichia coli chromosome usually initiates at a single origin (oriC) under control of DnaA. Two forks are established and move away in opposite directions. Replication is completed when these meet in a broadly defined terminus area half way around the circular chromosome. RecG appears to consolidate this arrangement by unwinding D-loops and R-loops that PriA might otherwise exploit to initiate replication at other sites. It has been suggested that without RecG such replication generates 3' flaps as the additional forks collide and displace nascent leading strands, providing yet more potential targets for PriA. Here we show that, to stay alive, cells must have either RecG or a 3' single-stranded DNA (ssDNA) exonuclease, which can be exonuclease I, exonuclease VII, or SbcCD. Cells lacking all three nucleases are inviable without RecG. They also need RecA recombinase and a Holliday junction resolvase to survive rapid growth, but SOS induction, although elevated, is not required. Additional requirements for Rep and UvrD are identified and linked with defects in DNA mismatch repair and with the ability to cope with conflicts between replication and transcription, respectively. Eliminating PriA helicase activity removes the requirement for RecG. The data are consistent with RecG and ssDNA exonucleases acting to limit PriA-mediated re-replication of the chromosome and the consequent generation of linear DNA branches that provoke recombination and delay chromosome segregation.
Assuntos
DNA Helicases/metabolismo , Replicação do DNA , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Exodesoxirribonucleases/metabolismo , Cromossomos Bacterianos/metabolismo , DNA Helicases/genética , Reparo de Erro de Pareamento de DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/citologia , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Exonucleases/metabolismo , Resolvases de Junção Holliday/genética , Resolvases de Junção Holliday/metabolismo , Recombinação GenéticaRESUMO
RuvABC and RecG are thought to provide alternative pathways for the late stages of recombination in Escherichia coli. Inactivation of both blocks the recovery of recombinants in genetic crosses. RuvABC resolves Holliday junctions, with RuvAB driving branch migration and RuvC catalyzing junction cleavage. RecG also drives branch migration, but no nuclease has been identified that might act with RecG to cleave junctions, apart from RusA, which is not normally expressed. We searched for an alternative nuclease using a synthetic lethality assay to screen for mutations causing inviability in the absence of RuvC, on the premise that a strain without any ability to cut junctions might be inviable. All the mutations identified mapped to polA, dam, or uvrD. None of these genes encodes a nuclease that cleaves Holliday junctions. Probing the reason for the inviability using the RusA Holliday junction resolvase provided strong evidence in each case that the RecG pathway is very ineffective at removing junctions and indicated that a nuclease component most probably does not exist. It also revealed new suppressors of recG, which were located to the ssb gene. Taken together with the results from the synthetic lethality assays, the properties of the mutant SSB proteins provide evidence that, rather than promoting recombination, a major function of RecG is to curb potentially pathological replication initiated via PriA protein at sites remote from oriC.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Resolvases de Junção Holliday/metabolismo , Recombinação Genética/genética , Substituição de Aminoácidos/genética , Proteínas de Bactérias/metabolismo , Cromossomos Bacterianos/metabolismo , DNA Helicases/metabolismo , Reparo do DNA , Replicação do DNA , DNA Bacteriano/metabolismo , Endodesoxirribonucleases/metabolismo , Escherichia coli/citologia , Viabilidade Microbiana , Modelos Biológicos , Supressão Genética/genéticaRESUMO
The rescue of replication forks stalled on the template DNA was investigated using an assay for synthetic lethality that provides a visual readout of cell viability and permits investigation of why certain mutations are lethal when combined. The results presented show that RecA and other recombination proteins are often engaged during replication because RecA is present and provokes recombination rather than because recombination is necessary. This occurs particularly frequently in cells lacking the helicase activities of Rep and PriA. We propose that these two proteins normally limit the loading of RecA on ssDNA regions exposed on the leading strand template of damaged forks, and do so by unwinding the nascent lagging strand, thus facilitating reannealing of the parental strands. Gap closure followed by loading of the DnaB replicative helicase enables synthesis of the leading strand to continue. Without either activity, RecA loads more frequently on the DNA and drives fork reversal, which creates a chickenfoot structure and a requirement for other recombination proteins to re-establish a viable fork. The assay also reveals that stalled transcription complexes are common impediments to fork progression, and that damaged forks often reverse independently of RecA.
Assuntos
Adenosina Trifosfatases/metabolismo , DNA Helicases/metabolismo , Reparo do DNA , Replicação do DNA , Recombinases Rec A/metabolismo , Recombinação Genética , Adenosina Trifosfatases/genética , DNA Helicases/genética , DNA Bacteriano , Recombinases Rec A/genéticaRESUMO
RecG differs from most helicases acting on branched DNA in that it is thought to catalyze unwinding via translocation of a monomer on dsDNA, with a wedge domain facilitating strand separation. Conserved phenylalanines in the wedge are shown to be critical for DNA binding. When detached from the helicase domains, the wedge bound a Holliday junction with high affinity but failed to bind a replication fork structure. Further stabilizing contacts are identified in full-length RecG, which may explain fork binding. Detached from the wedge, the helicase region unwound junctions but had extremely low substrate affinity, arguing against the "classical inchworm" mode of translocation. We propose that the processivity of RecG on branched DNA substrates is dependent on the ability of the wedge to establish strong binding at the branch point. This keeps the helicase motor in contact with the substrate, enabling it to drive dsDNA translocation with high efficiency.
Assuntos
DNA , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Estrutura Terciária de Proteína , Sequência de Aminoácidos , DNA/química , DNA/metabolismo , DNA Cruciforme/química , DNA Cruciforme/metabolismo , Proteínas de Escherichia coli/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
Recent studies in Escherichia coli indicate that the interconversion of DNA replication fork and Holliday junction structures underpins chromosome duplication and helps secure faithful transmission of the genome from one generation to the next. It facilitates interplay between DNA replication, recombination and repair, and provides means to rescue replication forks stalled by lesions in or on the template DNA. Insight into how this interconversion may be catalysed has emerged from genetic, biochemical and structural studies of RecG protein, a member of superfamily 2 of DNA and RNA helicases. We describe how a single molecule of RecG might target a branched DNA structure and translocate a single duplex arm to drive branch migration of a Holliday junction, interconvert replication fork and Holliday junction structures and displace the invading strand from a D loop formed during recombination at a DNA end. We present genetic evidence suggesting how the latter activity may provide an efficient pathway for the repair of DNA double-strand breaks that avoids crossing over, thus facilitating chromosome segregation at cell division.
Assuntos
Segregação de Cromossomos/fisiologia , DNA Helicases/química , Reparo do DNA/fisiologia , Replicação do DNA/fisiologia , Proteínas de Escherichia coli/química , Recombinação Genética/fisiologia , DNA/metabolismo , DNA Helicases/metabolismo , DNA Cruciforme/metabolismo , Escherichia coli , Proteínas de Escherichia coli/metabolismoRESUMO
RecG protein differs from other helicases analysed to atomic resolution in that it mediates strand separation via translocation on double-stranded (ds) rather than single-stranded (ss) DNA. We describe a highly conserved helical hairpin motif in RecG and show it to be important for helicase activity. It places two arginines (R609 and R630) in opposing positions within the component helices where they are stabilized by a network of hydrogen bonds involving a glutamate from helicase motif VI. We suggest that disruption of this feature, triggered by ATP hydrolysis, moves an adjacent loop structure in the dsDNA-binding channel and that a swinging arm motion of this loop drives translocation. Substitutions that reverse the charge at R609 or R630 reduce DNA unwinding and ATPase activities, and increase dsDNA binding, but do not affect branched DNA binding. Sequences forming the helical hairpin and loop structures are highly conserved in Mfd protein, a transcription-coupled DNA repair factor that also translocates on dsDNA. The possibility of type I restriction enzymes and chromatin-remodelling factors using similar structures to drive translocation on dsDNA is discussed.