RESUMO
Recent studies of bacterial DNA replication have led to a picture of the replisome as an entity that freely exchanges DNA polymerases and displays intermittent coupling between the helicase and polymerase(s). Challenging the textbook model of the polymerase holoenzyme acting as a stable complex coordinating the replisome, these observations suggest a role of the helicase as the central organizing hub. We show here that the molecular origin of this newly found plasticity lies in the 500-fold increase in strength of the interaction between the polymerase holoenzyme and the replicative helicase upon association of the primase with the replisome. By combining in vitro ensemble-averaged and single-molecule assays, we demonstrate that this conformational switch operates during replication and promotes recruitment of multiple holoenzymes at the fork. Our observations provide a molecular mechanism for polymerase exchange and offer a revised model for the replication reaction that emphasizes its stochasticity.
Assuntos
DNA Primase/metabolismo , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DnaB Helicases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Holoenzimas/química , DNA Primase/genética , DNA Bacteriano , DNA Polimerase Dirigida por DNA/genética , DnaB Helicases/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Holoenzimas/genética , Holoenzimas/metabolismo , Conformação Molecular , Ligação Proteica , Conformação ProteicaRESUMO
CRISPR-Cas immunity protects prokaryotes against invading genetic elements1. It uses the highly conserved Cas1-Cas2 complex to establish inheritable memory (spacers)2-5. How Cas1-Cas2 acquires spacers from foreign DNA fragments (prespacers) and integrates them into the CRISPR locus in the correct orientation is unclear6,7. Here, using the high spatiotemporal resolution of single-molecule fluorescence, we show that Cas1-Cas2 selects precursors of prespacers from DNA in various forms-including single-stranded DNA and partial duplexes-in a manner that depends on both the length of the DNA strand and the presence of a protospacer adjacent motif (PAM) sequence. We also identify DnaQ exonucleases as enzymes that process the Cas1-Cas2-loaded prespacer precursors into mature prespacers of a suitable size for integration. Cas1-Cas2 protects the PAM sequence from maturation, which results in the production of asymmetrically trimmed prespacers and the subsequent integration of spacers in the correct orientation. Our results demonstrate the kinetic coordination of prespacer precursor selection and PAM trimming, providing insight into the mechanisms that underlie the integration of functional spacers in the CRISPR loci.
Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA de Cadeia Simples/genética , Edição de Genes/métodos , Pareamento de Bases , DNA de Cadeia Simples/metabolismo , Exodesoxirribonuclease V/metabolismo , Exonucleases/metabolismo , Fluorescência , Cinética , Recombinação Genética/genética , Fatores de TempoRESUMO
Genome duplication occurs while the template DNA is bound by numerous DNA-binding proteins. Each of these proteins act as potential roadblocks to the replication fork and can have deleterious effects on cells. In Escherichia coli, these roadblocks are displaced by the accessory helicase Rep, a DNA translocase and helicase that interacts with the replisome. The mechanistic details underlying the coordination with replication and roadblock removal by Rep remain poorly understood. Through real-time fluorescence imaging of the DNA produced by individual E. coli replisomes and the simultaneous visualization of fluorescently-labeled Rep, we show that Rep continually surveils elongating replisomes. We found that this association of Rep with the replisome is stochastic and occurs independently of whether the fork is stalled or not. Further, we visualize the efficient rescue of stalled replication forks by directly imaging individual Rep molecules as they remove a model protein roadblock, dCas9, from the template DNA. Using roadblocks of varying DNA-binding stabilities, we conclude that continuation of synthesis is the rate-limiting step of stalled replication rescue.
Assuntos
DNA Helicases , Proteínas de Escherichia coli , DNA/metabolismo , DNA Helicases/química , Replicação do DNA , Escherichia coli/enzimologia , Proteínas de Escherichia coli/químicaRESUMO
Elongation by RNA polymerase is dynamically modulated by accessory factors. The transcription-repair coupling factor (TRCF) recognizes paused/stalled RNAPs and either rescues transcription or initiates transcription termination. Precisely how TRCFs choose to execute either outcome remains unclear. With Escherichia coli as a model, we used single-molecule assays to study dynamic modulation of elongation by Mfd, the bacterial TRCF. We found that nucleotide-bound Mfd converts the elongation complex (EC) into a catalytically poised state, presenting the EC with an opportunity to restart transcription. After long-lived residence in this catalytically poised state, ATP hydrolysis by Mfd remodels the EC through an irreversible process leading to loss of the RNA transcript. Further, biophysical studies revealed that the motor domain of Mfd binds and partially melts DNA containing a template strand overhang. The results explain pathway choice determining the fate of the EC and provide a molecular mechanism for transcription modulation by TRCF.
Assuntos
Proteínas de Bactérias , Reparo do DNA , Escherichia coli , Fatores de Transcrição , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
In Escherichia coli, the DnaB helicase forms the basis for the assembly of the DNA replication complex. The stability of DnaB at the replication fork is likely important for successful replication initiation and progression. Single-molecule experiments have significantly changed the classical model of highly stable replication machines by showing that components exchange with free molecules from the environment. However, due to technical limitations, accurate assessments of DnaB stability in the context of replication are lacking. Using in vitro fluorescence single-molecule imaging, we visualise DnaB loaded on forked DNA templates. That these helicases are highly stable at replication forks, indicated by their observed dwell time of â¼30 min. Addition of the remaining replication factors results in a single DnaB helicase integrated as part of an active replisome. In contrast to the dynamic behaviour of other replisome components, DnaB is maintained within the replisome for the entirety of the replication process. Interestingly, we observe a transient interaction of additional helicases with the replication fork. This interaction is dependent on the τ subunit of the clamp-loader complex. Collectively, our single-molecule observations solidify the role of the DnaB helicase as the stable anchor of the replisome, but also reveal its capacity for dynamic interactions.
Assuntos
Replicação do DNA , DnaB Helicases/metabolismo , DNA Polimerase Dirigida por DNA , Escherichia coli/genética , Complexos Multienzimáticos , Imagem Individual de MoléculaRESUMO
Bacterial single-stranded DNA-binding proteins (SSBs) bind single-stranded DNA and help to recruit heterologous proteins to their sites of action. SSBs perform these essential functions through a modular structural architecture: the N-terminal domain comprises a DNA binding/tetramerization element whereas the C-terminus forms an intrinsically disordered linker (IDL) capped by a protein-interacting SSB-Ct motif. Here we examine the activities of SSB-IDL fusion proteins in which fluorescent domains are inserted within the IDL of Escherichia coli SSB. The SSB-IDL fusions maintain DNA and protein binding activities in vitro, although cooperative DNA binding is impaired. In contrast, an SSB variant with a fluorescent protein attached directly to the C-terminus that is similar to fusions used in previous studies displayed dysfunctional protein interaction activity. The SSB-IDL fusions are readily visualized in single-molecule DNA replication reactions. Escherichia coli strains in which wildtype SSB is replaced by SSB-IDL fusions are viable and display normal growth rates and fitness. The SSB-IDL fusions form detectible SSB foci in cells with frequencies mirroring previously examined fluorescent DNA replication fusion proteins. Cells expressing SSB-IDL fusions are sensitized to some DNA damaging agents. The results highlight the utility of SSB-IDL fusions for biochemical and cellular studies of genome maintenance reactions.
Assuntos
Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/química , Fluorescência , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/química , Dano ao DNA , Reparo do DNA , Replicação do DNA , DNA de Cadeia Simples/química , Escherichia coli/citologia , Escherichia coli/genética , Escherichia coli/metabolismo , Genoma Bacteriano , Proteínas Intrinsicamente Desordenadas/química , Ligação Proteica , Resposta SOS em GenéticaRESUMO
Several functions have been proposed for the Escherichia coli DNA polymerase IV (pol IV). Although much research has focused on a potential role for pol IV in assisting pol III replisomes in the bypass of lesions, pol IV is rarely found at the replication fork in vivo. Pol IV is expressed at increased levels in E. coli cells exposed to exogenous DNA damaging agents, including many commonly used antibiotics. Here we present live-cell single-molecule microscopy measurements indicating that double-strand breaks induced by antibiotics strongly stimulate pol IV activity. Exposure to the antibiotics ciprofloxacin and trimethoprim leads to the formation of double strand breaks in E. coli cells. RecA and pol IV foci increase after treatment and exhibit strong colocalization. The induction of the SOS response, the appearance of RecA foci, the appearance of pol IV foci and RecA-pol IV colocalization are all dependent on RecB function. The positioning of pol IV foci likely reflects a physical interaction with the RecA* nucleoprotein filaments that has been detected previously in vitro. Our observations provide an in vivo substantiation of a direct role for pol IV in double strand break repair in cells treated with double strand break-inducing antibiotics.
Assuntos
Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Polimerase beta/ultraestrutura , Proteínas de Ligação a DNA/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/ultraestrutura , Exodesoxirribonuclease V/ultraestrutura , Recombinases Rec A/genética , Ciprofloxacina/farmacologia , Dano ao DNA/efeitos dos fármacos , DNA Polimerase beta/genética , Reparo do DNA/genética , Replicação do DNA/genética , Escherichia coli/genética , Escherichia coli/ultraestrutura , Exodesoxirribonuclease V/genética , Imagem Individual de MoléculaRESUMO
DNA lesions stall the replisome and proper resolution of these obstructions is critical for genome stability. Replisomes can directly replicate past a lesion by error-prone translesion synthesis. Alternatively, replisomes can reprime DNA synthesis downstream of the lesion, creating a single-stranded DNA gap that is repaired primarily in an error-free, homology-directed manner. Here we demonstrate how structural changes within the Escherichia coli replisome determine the resolution pathway of lesion-stalled replisomes. This pathway selection is controlled by a dynamic interaction between the proofreading subunit of the replicative polymerase and the processivity clamp, which sets a kinetic barrier to restrict access of translesion synthesis (TLS) polymerases to the primer/template junction. Failure of TLS polymerases to overcome this barrier leads to repriming, which competes kinetically with TLS. Our results demonstrate that independent of its exonuclease activity, the proofreading subunit of the replisome acts as a gatekeeper and influences replication fidelity during the resolution of lesion-stalled replisomes.
Assuntos
Dano ao DNA/genética , Reparo do DNA/genética , Replicação do DNA/genética , DNA Bacteriano , DNA Polimerase Dirigida por DNA , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMO
In all domains of life, DNA synthesis occurs bidirectionally from replication origins. Despite variable rates of replication fork progression, fork convergence often occurs at specific sites. Escherichia coli sets a 'replication fork trap' that allows the first arriving fork to enter but not to leave the terminus region. The trap is set by oppositely oriented Tus-bound Ter sites that block forks on approach from only one direction. However, the efficiency of fork blockage by Tus-Ter does not exceed 50% in vivo despite its apparent ability to almost permanently arrest replication forks in vitro. Here we use data from single-molecule DNA replication assays and structural studies to show that both polarity and fork-arrest efficiency are determined by a competition between rates of Tus displacement and rearrangement of Tus-Ter interactions that leads to blockage of slower moving replisomes by two distinct mechanisms. To our knowledge this is the first example where intrinsic differences in rates of individual replisomes have different biological outcomes.
Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Complexos Multienzimáticos/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Sequência de Bases , Ligação Competitiva , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , Cristalografia por Raios X , DNA Polimerase Dirigida por DNA/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Cinética , Modelos Biológicos , Modelos Moleculares , Movimento , Complexos Multienzimáticos/química , Conformação Proteica , Ressonância de Plasmônio de Superfície , Fatores de TempoRESUMO
Single-stranded DNA-binding proteins (SSBs) support DNA replication by protecting single-stranded DNA from nucleolytic attack, preventing intra-strand pairing events and playing many other regulatory roles within the replisome. Recent developments in single-molecule approaches have led to a revised picture of the replisome that is much more complex in how it retains or recycles protein components. Here, we visualize how an in vitro reconstituted Escherichia coli replisome recruits SSB by relying on two different molecular mechanisms. Not only does it recruit new SSB molecules from solution to coat newly formed single-stranded DNA on the lagging strand, but it also internally recycles SSB from one Okazaki fragment to the next. We show that this internal transfer mechanism is balanced against recruitment from solution in a manner that is concentration dependent. By visualizing SSB dynamics in live cells, we show that both internal transfer and external exchange mechanisms are physiologically relevant.
Assuntos
Replicação do DNA , DNA Bacteriano/genética , DNA de Cadeia Simples/genética , Escherichia coli/genética , DNA/genética , DNA/metabolismo , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , DNA Primase/genética , DNA Primase/metabolismo , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , DnaB Helicases/genética , DnaB Helicases/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Imagem com Lapso de TempoRESUMO
Synchronizing the convergence of the two-oppositely moving DNA replication machineries at specific termination sites is a tightly coordinated process in bacteria. In Escherichia coli, a "replication fork trap" - found within a chromosomal region where forks are allowed to enter but not leave - is set by the protein-DNA roadblock Tus-Ter. The exact sequence of events by which Tus-Ter blocks replisomes approaching from one direction but not the other has been the subject of controversy for many decades. Specific protein-protein interactions between the nonpermissive face of Tus and the approaching helicase were challenged by biochemical and structural studies. These studies show that it is the helicase-induced strand separation that triggers the formation of new Tus-Ter interactions at the nonpermissive face - interactions that result in a highly stable "locked" complex. This controversy recently gained renewed attention as three single-molecule-based studies scrutinized this elusive Tus-Ter mechanism - leading to new findings and refinement of existing models, but also generating new questions. Here, we discuss and compare the findings of each of the single-molecule studies to find their common ground, pinpoint the crucial differences that remain, and push the understanding of this bipartite DNA-protein system further.
Assuntos
Replicação do DNA , DNA Bacteriano/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Bactérias/química , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cromossomos Bacterianos/química , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Modelos Moleculares , Mapas de Interação de ProteínasRESUMO
Rolling-circle DNA amplification is a powerful tool employed in biotechnology to produce large from small amounts of DNA. This mode of DNA replication proceeds via a DNA topology that resembles a replication fork, thus also providing experimental access to the molecular mechanisms of DNA replication. However, conventional templates do not allow controlled access to multiple fork topologies, which is an important factor in mechanistic studies. Here we present the design and production of a rolling-circle substrate with a tunable length of both the gap and the overhang, and we show its application to the bacterial DNA-replication reaction.
Assuntos
Replicação do DNA/fisiologia , DNA Bacteriano/biossíntese , DNA Circular/biossíntese , Escherichia coli/química , Técnicas de Amplificação de Ácido Nucleico , DNA Bacteriano/química , DNA Circular/química , Escherichia coli/citologia , Conformação de Ácido Nucleico , Moldes GenéticosRESUMO
Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of serious hospital-acquired infections and is responsible for significant morbidity and mortality in residential care facilities. New agents against MRSA are needed to combat rising resistance to current antibiotics. We recently reported 5-hydroxy-3-methyl-1-phenyl-1H-pyrazole-4-carbodithioate (HMPC) as a new bacteriostatic agent against MRSA that appears to act via a novel mechanism. Here, twenty nine analogs of HMPC were synthesized, their anti-MRSA structure-activity relationships evaluated and selectivity versus human HKC-8 cells determined. Minimum inhibitory concentrations (MIC) ranged from 0.5 to 64⯵g/mL and up to 16-fold selectivity was achieved. The 4-carbodithioate function was found to be essential for activity but non-specific reactivity was ruled out as a contributor to antibacterial action. The study supports further work aimed at elucidating the molecular targets of this interesting new class of anti-MRSA agents.
Assuntos
Antibacterianos/química , Pirazóis/química , Tiocarbamatos/química , Tiocarbamatos/farmacologia , Antibacterianos/síntese química , Antibacterianos/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Pirazóis/síntese química , Pirazóis/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-Atividade , Tiocarbamatos/síntese químicaRESUMO
Escherichia coli has three DNA polymerases implicated in the bypass of DNA damage, a process called translesion synthesis (TLS) that alleviates replication stalling. Although these polymerases are specialized for different DNA lesions, it is unclear if they interact differently with the replication machinery. Of the three, DNA polymerase (Pol) II remains the most enigmatic. Here we report a stable ternary complex of Pol II, the replicative polymerase Pol III core complex and the dimeric processivity clamp, ß. Single-molecule experiments reveal that the interactions of Pol II and Pol III with ß allow for rapid exchange during DNA synthesis. As with another TLS polymerase, Pol IV, increasing concentrations of Pol II displace the Pol III core during DNA synthesis in a minimal reconstitution of primer extension. However, in contrast to Pol IV, Pol II is inefficient at disrupting rolling-circle synthesis by the fully reconstituted Pol III replisome. Together, these data suggest a ß-mediated mechanism of exchange between Pol II and Pol III that occurs outside the replication fork.
Assuntos
DNA Polimerase III/genética , DNA Polimerase II/genética , DNA Polimerase beta/genética , DNA/biossíntese , DNA/genética , Dano ao DNA/genética , DNA Polimerase II/química , DNA Polimerase III/química , DNA Polimerase beta/química , Reparo do DNA/genética , Replicação do DNA/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Estrutura Terciária de ProteínaRESUMO
Processive DNA synthesis by the αεθ core of the Escherichia coli Pol III replicase requires it to be bound to the ß2 clamp via a site in the α polymerase subunit. How the ε proofreading exonuclease subunit influences DNA synthesis by α was not previously understood. In this work, bulk assays of DNA replication were used to uncover a non-proofreading activity of ε. Combination of mutagenesis with biophysical studies and single-molecule leading-strand replication assays traced this activity to a novel ß-binding site in ε that, in conjunction with the site in α, maintains a closed state of the αεθ-ß2 replicase in the polymerization mode of DNA synthesis. The ε-ß interaction, selected during evolution to be weak and thus suited for transient disruption to enable access of alternate polymerases and other clamp binding proteins, therefore makes an important contribution to the network of protein-protein interactions that finely tune stability of the replicase on the DNA template in its various conformational states.
Assuntos
DNA Polimerase III/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Replicação do DNA/genética , Replicação do DNA/fisiologia , DNA de Cadeia Simples/biossíntese , DNA de Cadeia Simples/metabolismo , Estabilidade Enzimática/genética , Escherichia coli/genética , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica/fisiologia , Multimerização Proteica/genética , Multimerização Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , Homologia de Sequência de AminoácidosRESUMO
The bidirectional replication of a circular chromosome by many bacteria necessitates proper termination to avoid the head-on collision of the opposing replisomes. In Escherichia coli, replisome progression beyond the termination site is prevented by Tus proteins bound to asymmetric Ter sites. Structural evidence indicates that strand separation on the blocking (nonpermissive) side of Tus-Ter triggers roadblock formation, but biochemical evidence also suggests roles for protein-protein interactions. Here DNA unzipping experiments demonstrate that nonpermissively oriented Tus-Ter forms a tight lock in the absence of replicative proteins, whereas permissively oriented Tus-Ter allows nearly unhindered strand separation. Quantifying the lock strength reveals the existence of several intermediate lock states that are impacted by mutations in the lock domain but not by mutations in the DNA-binding domain. Lock formation is highly specific and exceeds reported in vivo efficiencies. We postulate that protein-protein interactions may actually hinder, rather than promote, proper lock formation.
Assuntos
Replicação do DNA , DNA Bacteriano/metabolismo , DNA Circular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Sequência de Bases , Sítios de Ligação , Cromossomos Bacterianos/química , Cromossomos Bacterianos/metabolismo , DNA Bacteriano/química , DNA Circular/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
The Escherichia coli replication terminator protein (Tus) binds to Ter sequences to block replication forks approaching from one direction. Here, we used single molecule and transient state kinetics to study responses of the heterologous phage T7 replisome to the Tus-Ter complex. The T7 replisome was arrested at the non-permissive end of Tus-Ter in a manner that is explained by a composite mousetrap and dynamic clamp model. An unpaired C(6) that forms a lock by binding into the cytosine binding pocket of Tus was most effective in arresting the replisome and mutation of C(6) removed the barrier. Isolated helicase was also blocked at the non-permissive end, but unexpectedly the isolated polymerase was not, unless C(6) was unpaired. Instead, the polymerase was blocked at the permissive end. This indicates that the Tus-Ter mechanism is sensitive to the translocation polarity of the DNA motor. The polymerase tracking along the template strand traps the C(6) to prevent lock formation; the helicase tracking along the other strand traps the complementary G(6) to aid lock formation. Our results are consistent with the model where strand separation by the helicase unpairs the GC(6) base pair and triggers lock formation immediately before the polymerase can sequester the C(6) base.
Assuntos
Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Pareamento de Bases , DNA/biossíntese , DNA/química , DNA Helicases/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Modelos GenéticosRESUMO
Translesion synthesis (TLS) by Y-family DNA polymerases alleviates replication stalling at DNA damage. Ring-shaped processivity clamps play a critical but ill-defined role in mediating exchange between Y-family and replicative polymerases during TLS. By reconstituting TLS at the single-molecule level, we show that the Escherichia coli ß clamp can simultaneously bind the replicative polymerase (Pol) III and the conserved Y-family Pol IV, enabling exchange of the two polymerases and rapid bypass of a Pol IV cognate lesion. Furthermore, we find that a secondary contact between Pol IV and ß limits Pol IV synthesis under normal conditions but facilitates Pol III displacement from the primer terminus following Pol IV induction during the SOS DNA damage response. These results support a role for secondary polymerase clamp interactions in regulating exchange and establishing a polymerase hierarchy.
Assuntos
DNA Polimerase III/metabolismo , DNA Polimerase beta/metabolismo , DNA/metabolismo , Modelos Genéticos , Resposta SOS em Genética/fisiologia , Escherichia coli , Técnicas Analíticas Microfluídicas , Ligação Proteica , Estatísticas não ParamétricasRESUMO
During DNA replication, repetitive synthesis of discrete Okazaki fragments requires mechanisms that guarantee DNA polymerase, clamp, and primase proteins are present for every cycle. In Escherichia coli, this process proceeds through transfer of the lagging-strand polymerase from the ß sliding clamp left at a completed Okazaki fragment to a clamp assembled on a new RNA primer. These lagging-strand clamps are thought to be bound by the replisome from solution and loaded a new for every fragment. Here, we discuss a surprising, alternative lagging-strand synthesis mechanism: efficient replication in the absence of any clamps other than those assembled with the replisome. Using single-molecule experiments, we show that replication complexes pre-assembled on DNA support synthesis of multiple Okazaki fragments in the absence of excess ß clamps. The processivity of these replisomes, but not the number of synthesized Okazaki fragments, is dependent on the frequency of RNA-primer synthesis. These results broaden our understanding of lagging-strand synthesis and emphasize the stability of the replisome to continue synthesis without new clamps.
Assuntos
DNA Polimerase III/deficiência , Replicação do DNA/fisiologia , DNA/biossíntese , Escherichia coli/fisiologia , Modelos Biológicos , Microscopia Eletrônica , Microscopia de FluorescênciaRESUMO
A complex of the three (αεθ) core subunits and the ß2 sliding clamp is responsible for DNA synthesis by Pol III, the Escherichia coli chromosomal DNA replicase. The 1.7 Å crystal structure of a complex between the PHP domain of α (polymerase) and the C-terminal segment of ε (proofreading exonuclease) subunits shows that ε is attached to α at a site far from the polymerase active site. Both α and ε contain clamp-binding motifs (CBMs) that interact simultaneously with ß2 in the polymerization mode of DNA replication by Pol III. Strengthening of both CBMs enables isolation of stable αεθ:ß2 complexes. Nuclear magnetic resonance experiments with reconstituted αεθ:ß2 demonstrate retention of high mobility of a segment of 22 residues in the linker that connects the exonuclease domain of ε with its α-binding segment. In spite of this, small-angle X-ray scattering data show that the isolated complex with strengthened CBMs has a compact, but still flexible, structure. Photo-crosslinking with p-benzoyl-L-phenylalanine incorporated at different sites in the α-PHP domain confirm the conformational variability of the tether. Structural models of the αεθ:ß2 replicase complex with primer-template DNA combine all available structural data.