RESUMO
DNA replication must cope with nucleoprotein barriers that impair efficient replisome translocation. Biochemical and genetic studies indicate accessory helicases play essential roles in replication in the presence of nucleoprotein barriers, but how they operate inside the cell is unclear. With high-speed single-molecule microscopy we observed genomically-encoded fluorescent constructs of the accessory helicase Rep and core replisome protein DnaQ in live Escherichia coli cells. We demonstrate that Rep colocalizes with 70% of replication forks, with a hexameric stoichiometry, indicating maximal occupancy of the single DnaB hexamer. Rep associates dynamically with the replisome with an average dwell time of 6.5 ms dependent on ATP hydrolysis, indicating rapid binding then translocation away from the fork. We also imaged PriC replication restart factor and observe Rep-replisome association is also dependent on PriC. Our findings suggest two Rep-replisome populations in vivo: one continually associating with DnaB then translocating away to aid nucleoprotein barrier removal ahead of the fork, another assisting PriC-dependent reloading of DnaB if replisome progression fails. These findings reveal how a single helicase at the replisome provides two independent ways of underpinning replication of protein-bound DNA, a problem all organisms face as they replicate their genomes.
Assuntos
DNA Helicases/metabolismo , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Complexos Multienzimáticos/metabolismo , Trifosfato de Adenosina/metabolismo , DNA Helicases/química , DNA Polimerase III/metabolismo , Proteínas de Escherichia coli/química , Domínios e Motivos de Interação entre Proteínas , Imagem Individual de MoléculaRESUMO
Bacterial genome duplication and transcription require simultaneous access to the same DNA template. Conflicts between the replisome and transcription machinery can lead to interruption of DNA replication and loss of genome stability. Pausing, stalling and backtracking of transcribing RNA polymerases add to this problem and present barriers to replisomes. Accessory helicases promote fork movement through nucleoprotein barriers and exist in viruses, bacteria and eukaryotes. Here, we show that stalled Escherichia coli transcription elongation complexes block reconstituted replisomes. This physiologically relevant block can be alleviated by the accessory helicase Rep or UvrD, resulting in the formation of full-length replication products. Accessory helicase action during replication-transcription collisions therefore promotes continued replication without leaving gaps in the DNA. In contrast, DinG does not promote replisome movement through stalled transcription complexes in vitro. However, our data demonstrate that DinG operates indirectly in vivo to reduce conflicts between replication and transcription. These results suggest that Rep and UvrD helicases operate on DNA at the replication fork whereas DinG helicase acts via a different mechanism.
Assuntos
DNA Helicases/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , DNA Helicases/genética , Reparo do DNA , Replicação do DNA , DNA Bacteriano/biossíntese , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala , Complexos Multienzimáticos/metabolismo , Transcrição GênicaRESUMO
Helicases catalyse DNA and RNA strand separation. Proteins bound to the nucleic acid must also be displaced in order to unwind DNA. This is exemplified by accessory helicases that clear protein barriers from DNA ahead of advancing replication forks. How helicases catalyse DNA unwinding is increasingly well understood but how protein displacement is achieved is unclear. Escherichia coli Rep accessory replicative helicase lacking one of its four subdomains, 2B, has been shown to be hyperactivated for DNA unwinding in vitro but we show here that RepΔ2B is, in contrast, deficient in displacing proteins from DNA. This defect correlates with an inability to promote replication of protein-bound DNA in vitro and lack of accessory helicase function in vivo. Defective protein displacement is manifested on double-stranded and single-stranded DNA. Thus binding and distortion of duplex DNA by the 2B subdomain ahead of the helicase is not the missing function responsible for this deficiency. These data demonstrate that protein displacement from DNA is not simply achieved by helicase translocation alone. They also imply that helicases may have evolved different specific features to optimise DNA unwinding and protein displacement, both of which are now recognised as key functions in all aspects of nucleic acid metabolism.
Assuntos
DNA Helicases/química , DNA Bacteriano/química , DNA de Cadeia Simples/química , DNA/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , DNA/genética , DNA/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , DNA Primase/genética , DNA Primase/metabolismo , Replicação do DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease EcoRI/genética , Desoxirribonuclease EcoRI/metabolismo , DnaB Helicases/genética , DnaB Helicases/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Modelos Moleculares , Conformação de Ácido Nucleico , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre ProteínasRESUMO
Cascade complexes underpin E. coli CRISPR-Cas immunity systems by stimulating 'adaptation' reactions that update immunity and by initiating 'interference' reactions that destroy invader DNA. Recognition of invader DNA in Cascade catalysed R-loops provokes DNA capture and its subsequent integration into CRISPR loci by Cas1 and Cas2. DNA capture processes are unclear but may involve RecG helicase, which stimulates adaptation during its role responding to genome instability. We show that Cascade is a potential source of genome instability because it blocks DNA replication and that RecG helicase alleviates this by dissociating Cascade. This highlights how integrating in vitro CRISPR-Cas interference and adaptation reactions with DNA replication and repair reactions will help to determine precise mechanisms underpinning prokaryotic adaptive immunity.
Assuntos
Sistemas CRISPR-Cas/genética , Reparo do DNA , Replicação do DNA , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Plasmídeos/genéticaRESUMO
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
The PcrA/UvrD helicase functions in multiple pathways that promote bacterial genome stability including the suppression of conflicts between replication and transcription and facilitating the repair of transcribed DNA. The reported ability of PcrA/UvrD to bind and backtrack RNA polymerase (1,2) might be relevant to these functions, but the structural basis for this activity is poorly understood. In this work, we define a minimal RNA polymerase interaction domain in PcrA, and report its crystal structure at 1.5 Å resolution. The domain adopts a Tudor-like fold that is similar to other RNA polymerase interaction domains, including that of the prototype transcription-repair coupling factor Mfd. Removal or mutation of the interaction domain reduces the ability of PcrA/UvrD to interact with and to remodel RNA polymerase complexes in vitro. The implications of this work for our understanding of the role of PcrA/UvrD at the interface of DNA replication, transcription and repair are discussed.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Aminoácidos/química , Geobacillus stearothermophilus/enzimologia , Modelos Moleculares , Ligação Proteica , Elongação da Transcrição Genética , Domínio TudorRESUMO
How DNA metabolism is adapted to survival of organisms such as the bacterium Photobacterium profundum SS9 at high pressure is unknown. Previously, a high pressure-sensitive P. profundum SS9 transposon mutant (FL31) was identified, with an insertion in a putative rctB gene. The Vibrio cholerae RctB protein is essential for replication initiation at the origin of chromosome II, oriCII. Using a plasmid-based system in E. coli we have identified the replication origin of chromosome II from P. profundum SS9 and have shown that the putative rctB gene, disrupted in FL31, is essential for oriCII function. Moreover, we found that a region corresponding to the V. cholerae oriCII incompatibility region (incII) exerts an inhibitory effect on P. profundum oriCII. The truncated rctB gene in FL31 confers insensitivity to incII inhibition, indicating that the C-terminus of RctB is important for the negative regulation of replication. The RctB proteins of V. cholerae and P. profundum are partially interchangeable, but full functionality is achieved only with the cognate origin. Our findings provide the first characterization of the replication origin of chromosome II in a deep-sea bacterium.
Assuntos
Cromossomos Bacterianos/genética , Replicação do DNA , Photobacterium/genética , Origem de Replicação/genética , Adaptação Fisiológica/genética , Pressão Atmosférica , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cromossomos Bacterianos/metabolismo , DNA Helicases/química , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/genética , Genes Bacterianos/genética , Mutação , Photobacterium/crescimento & desenvolvimento , Photobacterium/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Transativadores/química , Transativadores/genética , Transativadores/metabolismo , Vibrio cholerae/genéticaRESUMO
DNA three-way junctions (3WJs) are branched structures that serve as important biological intermediates and as components in DNA nanostructures. We recently derived the global structure of a fully complementary 3WJ and found that it contained unpaired bases at the branchpoint, which is consistent with previous observations of branch flexibility and branchpoint reactivity. By combining high-resolution single-molecule Förster resonance energy transfer, molecular modeling, time-resolved ensemble fluorescence spectroscopy, and the first 19F nuclear magnetic resonance observations of fully complementary 3WJs, we now show that the 3WJ structure can adopt multiple distinct conformations depending upon the sequence at the branchpoint. A 3WJ with a GC-rich branchpoint adopts an open conformation with unpaired bases at the branch and at least one additional conformation with an increased number of base interactions at the branchpoint. This structural diversity has implications for branch interactions and processing in vivo and for technological applications.
Assuntos
DNA Complementar/química , DNA/química , Modelos Moleculares , Pareamento de Bases , DNA/metabolismo , DNA Complementar/metabolismo , Transferência Ressonante de Energia de Fluorescência , Sequência Rica em GC , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Imagem Individual de Molécula , Espectrometria de FluorescênciaRESUMO
Helicases are a subfamily of translocases that couple the directional translocation along a nucleic acid lattice to the separation of nucleic acid duplexes using the energy derived from nucleoside triphosphate hydrolysis. These enzymes perform essential functions in all aspects of nucleic acid metabolism by unwinding and remodelling DNA or RNA in DNA replication, repair, recombination, transcription and translation. Most classical biochemical studies assay the ability of these enzymes to separate naked nucleic acids. However, many different types of proteins form non-covalent interactions with nucleic acids in vivo and so the true substrates of helicases are protein-nucleic acid complexes rather than naked DNA and RNA. Studies over the last decade have revealed that bound proteins can have substantial inhibitory effects on the ability of helicases to unwind nucleic acids. Any analysis of helicase mechanisms in vitro must therefore consider helicase function within the context of nucleoprotein substrates rather than just DNA or RNA. Here we discuss how to analyse the impact of bound proteins on the ability of helicases to unwind DNA substrates in vitro.
Assuntos
DNA Helicases/genética , Replicação do DNA/genética , DNA de Cadeia Simples/genética , Nucleoproteínas/genética , Sequência de Bases/genética , DNA Helicases/química , DNA de Cadeia Simples/química , Hidrólise , Conformação de Ácido Nucleico , Nucleoproteínas/químicaRESUMO
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
BACKGROUND: Point-of-care (POC) CD4 testing increases patient accessibility to assessment of antiretroviral therapy eligibility. This review evaluates field performance in low and middle-income countries (LMICs) of currently available POC CD4 technologies. METHODS: Eight electronic databases were searched for field studies published between January 2005 and January 2015 of six POC CD4 platforms: PointCare NOW™, Alere Pima™ CD4, Daktari™ CD4 Counter, CyFlow® CD4 miniPOC, BD FACSPresto™, and MyT4™ CD4. Due to limited data availability, meta-analysis was conducted only for diagnostic performance of Pima at a threshold of 350 cells/µl, applying a bivariate multi-level random-effects modelling approach. A covariate extended model was also explored to test for difference in diagnostic performance between capillary and venous blood. RESULTS: Twenty seven studies were included. Published field study results were found for three of the six POC CD4 tests, 24 of which used Pima. For Pima, test failure rates varied from 2 to 23 % across study settings. Pooled sensitivity and specificity were 0.92 (95 % CI = 0.88-0.95) and 0.87 (95 % CI = 0.85-0.88) respectively. Diagnostic performance by blood sample type (venous vs. capillary) revealed non-significant differences in sensitivity (0.94 vs 0.89) and specificity (0.86 vs 0.87), respectively in the extended model (Wald χ2(2) = 4.77, p = 0.09). CONCLUSIONS: POC CD4 testing can provides reliable results for making treatment decision under field conditions in low-resource settings. The Pima test shows a good diagnostic performance at CD4 cut-off of 350 cells/µl. More data are required to evaluate performance of POC CD4 testing using venous versus capillary blood in LMICs which might otherwise influence clinical practice.
Assuntos
Contagem de Linfócito CD4/métodos , Sistemas Automatizados de Assistência Junto ao Leito , Países em Desenvolvimento , Infecções por HIV/sangue , Infecções por HIV/tratamento farmacológico , Recursos em Saúde , Humanos , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: CD4 testing is, and will remain an important part of HIV treatment and care in low and middle income countries (LMICs). We report the findings of a systematic review assessing acceptability and feasibility of POC CD4 testing in field settings. METHODS: Electronic databases were searched for studies published in English between 2005 and 2015 that describe POC CD4 platforms. Studies conducted in LMICs and under field conditions outside a laboratory environment were eligible. Qualitative and descriptive data analysis was used to present the findings. RESULTS: Twelve studies were included, 11 of which were conducted in sub-Saharan countries and used one POC CD4 test (The Alere Pima CD4). Patients reported positively regarding the implementation of POC CD4 testing at primary health care and community level with ≥90 % of patients accepting the test across various study settings. Health service providers expressed preference toward POC CD4 testing as it is easy-to-use, efficient and satisfied patients' needs to a greater extent as compared to conventional methods. However, operational challenges including preference toward venous blood rather than finger-prick sampling, frequent device failures and operator errors, quality of training for test operators and supervisors, and increased staff workload were also identified. CONCLUSIONS: POC CD4 testing seems acceptable and feasible in LIMCs under field conditions. Further studies using different POC CD4 tests available on the market are required to provide critical data to support countries in selection and implementation of appropriate POC CD4 technologies.
Assuntos
Contagem de Linfócito CD4 , Continuidade da Assistência ao Paciente , Infecções por HIV , Testes Imediatos , Adulto , Coleta de Amostras Sanguíneas , Países em Desenvolvimento , Falha de Equipamento , Feminino , Infecções por HIV/tratamento farmacológico , Humanos , Masculino , Pessoa de Meia-Idade , Satisfação Pessoal , Atenção Primária à SaúdeRESUMO
The method of action of many antibiotics is to interfere with DNA replication-quinolones trap DNA gyrase and topoisomerase proteins onto DNA while metronidazole causes single- and double-stranded breaks in DNA. To understand how bacteria respond to these drugs, it is important to understand the repair processes utilised when DNA replication is blocked. We have used tandem lac operators inserted into the chromosome bound by fluorescently labelled lac repressors as a model protein block to replication in E. coli. We have used dual-colour, alternating-laser, single-molecule narrowfield microscopy to quantify the amount of operator at the block and simultaneously image fluorescently labelled DNA polymerase. We anticipate use of this system as a quantitative platform to study replication stalling and repair proteins.
Assuntos
Antibacterianos/farmacologia , Replicação do DNA/efeitos dos fármacos , DNA Bacteriano/efeitos dos fármacos , Infecções por Escherichia coli/tratamento farmacológico , Escherichia coli/efeitos dos fármacos , Microscopia de Fluorescência , Imagem Molecular/métodos , Animais , DNA Bacteriano/biossíntese , DNA Bacteriano/genética , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Processamento de Imagem Assistida por Computador , Óperon Lac , Repressores Lac/genética , Repressores Lac/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismoRESUMO
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
DNA replication in all organisms must overcome nucleoprotein blocks to complete genome duplication. Accessory replicative helicases in Escherichia coli, Rep and UvrD, help remove these blocks and aid the re-initiation of replication. Mechanistic details of Rep function have emerged from recent live cell studies; however, the division of UvrD functions between its activities in DNA repair and role as an accessory helicase remain unclear in live cells. By integrating super-resolved single-molecule fluorescence microscopy with biochemical analysis, we find that UvrD self-associates into tetrameric assemblies and, unlike Rep, is not recruited to a specific replisome protein despite being found at approximately 80% of replication forks. Instead, its colocation with forks is likely due to the very high frequency of replication blocks composed of DNA-bound proteins, including RNA polymerase and factors involved in repairing DNA damage. Deleting rep and DNA repair factor genes mutS and uvrA, and inhibiting transcription through RNA polymerase mutation and antibiotic inhibition, indicates that the level of UvrD at the fork is dependent on UvrD's function. Our findings show that UvrD is recruited to sites of nucleoprotein blocks via different mechanisms to Rep and plays a multi-faceted role in ensuring successful DNA replication.
Assuntos
DNA Helicases , Replicação do DNA , Proteínas de Escherichia coli , Escherichia coli , DNA Helicases/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo , Nucleoproteínas/genética , Nucleoproteínas/metabolismoRESUMO
There is mounting evidence that there are frequent conflicts between complexes that replicate DNA and those that transcribe the same template, and that these conflicts lead to blockage of replication and genome instability. Such problems are perhaps best understood in bacteria, but it is becoming apparent that replicative barriers associated with transcription are a universal feature of life. This review summarizes what is currently known about how collisions between replisomes and transcription complexes are minimized and the mechanisms that help to resolve such collisions when they do occur. Although our understanding of these processes is still far from complete, a picture is emerging of a wide variety of different types of transcriptional blocks to replication that have resulted in a complex, overlapping system of mechanisms to avoid or tolerate such collisions.
Assuntos
Bactérias/genética , Replicação do DNA , Instabilidade Genômica , Transcrição Gênica , RNA Polimerases Dirigidas por DNA/metabolismo , Saccharomyces cerevisiae/genéticaRESUMO
If replication forks are perturbed, a multifaceted response including several DNA repair and cell cycle checkpoint pathways is activated to ensure faithful DNA replication. Here, we show that poly(ADP-ribose) polymerase 1 (PARP1) binds to and is activated by stalled replication forks that contain small gaps. PARP1 collaborates with Mre11 to promote replication fork restart after release from replication blocks, most likely by recruiting Mre11 to the replication fork to promote resection of DNA. Both PARP1 and PARP2 are required for hydroxyurea-induced homologous recombination to promote cell survival after replication blocks. Together, our data suggest that PARP1 and PARP2 detect disrupted replication forks and attract Mre11 for end processing that is required for subsequent recombination repair and restart of replication forks.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Recombinação Genética/fisiologia , Animais , Células Cultivadas , Cricetinae , Cricetulus , Reparo do DNA , ImunofluorescênciaRESUMO
Genome duplication requires not only unwinding of the template but also the displacement of proteins bound to the template, a function performed by replicative helicases located at the fork. However, accessory helicases are also needed since the replicative helicase stalls occasionally at nucleoprotein complexes. In Escherichia coli, the primary and accessory helicases DnaB and Rep translocate along the lagging and leading strand templates, respectively, interact physically and also display cooperativity in the unwinding of model forked DNA substrates. We demonstrate here that this cooperativity is displayed only by Rep and not by other tested helicases. ssDNA must be exposed on the leading strand template to elicit this cooperativity, indicating that forks blocked at protein-DNA complexes contain ssDNA ahead of the leading strand polymerase. However, stable Rep-DnaB complexes can form on linear as well as branched DNA, indicating that Rep has the capacity to interact with ssDNA on either the leading or the lagging strand template at forks. Inhibition of Rep binding to the lagging strand template by competition with SSB might therefore be critical in targeting accessory helicases to the leading strand template, indicating an important role for replisome architecture in promoting accessory helicase function at blocked replisomes.
Assuntos
DNA Helicases/metabolismo , DNA/metabolismo , DnaB Helicases/metabolismo , Proteínas de Escherichia coli/metabolismo , DNA de Cadeia Simples/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
Helicases are fundamental components of all replication complexes since unwinding of the double-stranded template to generate single-stranded DNA is essential to direct DNA synthesis by polymerases. However, helicases are also required in many other steps of DNA replication. Replicative helicases not only unwind the template DNA but also play key roles in regulating priming of DNA synthesis and coordination of leading and lagging strand DNA polymerases. Accessory helicases also aid replicative helicases in unwinding of the template strands in the presence of proteins bound to the DNA, minimising the risks posed by nucleoprotein complexes to continued fork movement. Helicases also play critical roles in Okazaki fragment processing in eukaryotes and may also be needed to minimise topological problems when replication forks converge. Thus fork movement, coordination of DNA synthesis, lagging strand maturation and termination of replication all depend on helicases. Moreover, if disaster strikes and a replication fork breaks down then reloading of the replication machinery is effected by helicases, at least in bacteria. This chapter describes how helicases function in these multiple steps at the fork and how DNA unwinding is coordinated with other catalytic processes to ensure efficient, high fidelity duplication of the genetic material in all organisms.