RESUMO
The eukaryotic replicative helicase CMG is a closed ring around double-stranded (ds)DNA at origins yet must transition to single-stranded (ss)DNA for helicase action. CMG must also handle repair intermediates, such as reversed forks that lack ssDNA. Here, using correlative single-molecule fluorescence and force microscopy, we show that CMG harbors a ssDNA gate that enables transitions between ss and dsDNA. When coupled to DNA polymerase, CMG remains on ssDNA, but when uncoupled, CMG employs this gate to traverse forked junctions onto dsDNA. Surprisingly, CMG undergoes rapid diffusion on dsDNA and can transition back onto ssDNA to nucleate a functional replisome. The gate-distinct from that between Mcm2/5 used for origin loading-is intrinsic to CMG; however, Mcm10 promotes strand passage by enhancing the affinity of CMG to DNA. This gating process may explain the dsDNA-to-ssDNA transition of CMG at origins and help preserve CMG on dsDNA during fork repair.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Manutenção de Minicromossomo/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , DNA/metabolismo , Replicação do DNA , DNA de Cadeia Simples/química , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Structural and biochemical studies have revealed the basic principles of how the replisome duplicates genomic DNA, but little is known about its dynamics during DNA replication. We reconstitute the 34 proteins needed to form the S. cerevisiae replisome and show how changing local concentrations of the key DNA polymerases tunes the ability of the complex to efficiently recycle these proteins or to dynamically exchange them. Particularly, we demonstrate redundancy of the Pol α-primase DNA polymerase activity in replication and show that Pol α-primase and the lagging-strand Pol δ can be re-used within the replisome to support the synthesis of large numbers of Okazaki fragments. This unexpected malleability of the replisome might allow it to deal with barriers and resource challenges during replication of large genomes.
Assuntos
DNA Polimerase III/genética , Replicação do DNA/genética , DNA/genética , Células Eucarióticas/fisiologia , DNA Polimerase I/genética , DNA Primase/genética , Saccharomyces cerevisiae/genéticaRESUMO
Efficient and faithful replication of the genome is essential to maintain genome stability. Replication is carried out by a multiprotein complex called the replisome, which encounters numerous obstacles to its progression. Failure to bypass these obstacles results in genome instability and may facilitate errors leading to disease. Cells use accessory helicases that help the replisome bypass difficult barriers. All eukaryotes contain the accessory helicase Pif1, which tracks in a 5'-3' direction on single-stranded DNA and plays a role in genome maintenance processes. Here, we reveal a previously unknown role for Pif1 in replication barrier bypass. We use an in vitro reconstituted Saccharomyces cerevisiae replisome to demonstrate that Pif1 enables the replisome to bypass an inactive (i.e., dead) Cas9 (dCas9) R-loop barrier. Interestingly, dCas9 R-loops targeted to either strand are bypassed with similar efficiency. Furthermore, we employed a single-molecule fluorescence visualization technique to show that Pif1 facilitates this bypass by enabling the simultaneous removal of the dCas9 protein and the R-loop. We propose that Pif1 is a general displacement helicase for replication bypass of both R-loops and protein blocks.
Assuntos
Replicação do DNA , DNA/genética , DNA/metabolismo , Estruturas R-Loop , Proteínas de Ligação a Telômeros/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , DNA/química , Edição de Genes , Modelos Biológicos , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , RNA Guia de CinetoplastídeosRESUMO
The AID/APOBEC enzymes deaminate cytosines in single-stranded DNA (ssDNA) and play key roles in innate and adaptive immunity. The resulting uracils cause mutations and strand breaks that inactivate viruses and diversify antibody repertoire. Mutational evidence suggests that two members of this family, APOBEC3A (A3A) and APOBEC3B, deaminate cytosines in the lagging-strand template during replication. To obtain direct evidence for the presence of these uracils, we engineered a protein that covalently links to DNA at uracils, UdgX, for mammalian expression and immunohistochemistry. We show that UdgX strongly prefers uracils in ssDNA over those in Uâ¢G or U:A pairs, and localizes to nuclei in a dispersed form. When A3A is expressed in these cells, UdgX tends to form foci. The treatment of cells with cisplatin, which blocks replication, causes a significant increase in UdgX foci. Furthermore, this protein- and hence the uracils created by A3A- colocalize with replication protein A (RPA), but not with A3A. Using purified proteins, we confirm that RPA inhibits A3A by binding ssDNA, but despite its overexpression following cisplatin treatment, RPA is unable to fully protect ssDNA created by cisplatin adducts. This suggests that cisplatin treatment of cells expressing APOBEC3A should cause accumulation of APOBEC signature mutations.
Assuntos
Citidina Desaminase/metabolismo , Citosina/metabolismo , Replicação do DNA , DNA/química , Proteínas/metabolismo , Proteína de Replicação A/metabolismo , Uracila-DNA Glicosidase/genética , Uracila/análise , Núcleo Celular/metabolismo , Cisplatino/farmacologia , Reagentes de Ligações Cruzadas/farmacologia , Citidina Desaminase/genética , DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Células HEK293 , Células HeLa , Humanos , Imidazóis/farmacologia , Engenharia de Proteínas , Proteínas/genética , Uracila/química , Uracila/metabolismoRESUMO
The eukaryotic genome is primarily replicated by two DNA polymerases, Pol ε and Pol δ, that function on the leading and lagging strands, respectively. Previous studies have established recruitment mechanisms whereby Cdc45-Mcm2-7-GINS (CMG) helicase binds Pol ε and tethers it to the leading strand, and PCNA (proliferating cell nuclear antigen) binds tightly to Pol δ and recruits it to the lagging strand. The current report identifies quality control mechanisms that exclude the improper polymerase from a particular strand. We find that the replication factor C (RFC) clamp loader specifically inhibits Pol ε on the lagging strand, and CMG protects Pol ε against RFC inhibition on the leading strand. Previous studies show that Pol δ is slow and distributive with CMG on the leading strand. However, Saccharomyces cerevisiae Pol δ-PCNA is a rapid and processive enzyme, suggesting that CMG may bind and alter Pol δ activity or position it on the lagging strand. Measurements of polymerase binding to CMG demonstrate Pol ε binds CMG with a Kd value of 12 nM, but Pol δ binding CMG is undetectable. Pol δ, like bacterial replicases, undergoes collision release upon completing replication, and we propose Pol δ-PCNA collides with the slower CMG, and in the absence of a stabilizing Pol δ-CMG interaction, the collision release process is triggered, ejecting Pol δ on the leading strand. Hence, by eviction of incorrect polymerases at the fork, the clamp machinery directs quality control on the lagging strand and CMG enforces quality control on the leading strand.
Assuntos
DNA Polimerase III/metabolismo , DNA Polimerase II/metabolismo , Replicação do DNA/genética , Células Eucarióticas/metabolismo , Proteínas de Manutenção de Minicromossomo/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Controle de Qualidade , RNA Polimerase Dependente de RNA/metabolismo , Proteína de Replicação C/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The replisome, the multiprotein system responsible for genome duplication, is a highly dynamic complex displaying a large number of different enzyme activities. Recently, the Saccharomyces cerevisiae minimal replication reaction has been successfully reconstituted in vitro. This provided an opportunity to uncover the enzymatic activities of many of the components in a eukaryotic system. Their dynamic behavior and interactions in the context of the replisome, however, remain unclear. We use a tethered-bead assay to provide real-time visualization of leading-strand synthesis by the S. cerevisiae replisome at the single-molecule level. The minimal reconstituted leading-strand replisome requires 24 proteins, forming the CMG helicase, the Pol ε DNA polymerase, the RFC clamp loader, the PCNA sliding clamp, and the RPA single-stranded DNA binding protein. We observe rates and product lengths similar to those obtained from ensemble biochemical experiments. At the single-molecule level, we probe the behavior of two components of the replication progression complex and characterize their interaction with active leading-strand replisomes. The Minichromosome maintenance protein 10 (Mcm10), an important player in CMG activation, increases the number of productive replication events in our assay. Furthermore, we show that the fork protection complex Mrc1-Tof1-Csm3 (MTC) enhances the rate of the leading-strand replisome threefold. The introduction of periods of fast replication by MTC leads to an average rate enhancement of a factor of 2, similar to observations in cellular studies. We observe that the MTC complex acts in a dynamic fashion with the moving replisome, leading to alternating phases of slow and fast replication.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Replicação do DNA/fisiologia , DNA Fúngico/biossíntese , Proteínas de Ligação a DNA/metabolismo , Proteínas de Manutenção de Minicromossomo/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Proteínas de Manutenção de Minicromossomo/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are routinely used to treat HIV-1 infection, yet their mechanism of action remains unclear despite intensive investigation. In this study, we developed complementary single-molecule fluorescence and ensemble fluorescence anisotropy approaches to discover how NNRTIs modulate the intra-molecular conformational changes and inter-molecular dynamics of RT-template/primer (T/P) and RT-T/P-dNTP complexes. We found that NNRTI binding to RT induces opening of the fingers and thumb subdomains, which increases the dynamic sliding motion of the enzyme on the T/P and reduces dNTP binding affinity. Further, efavirenz promotes formation of the E138-K101 salt bridge between the p51 and p66 subunits of RT, which contributes to opening of the thumb/fingers subdomains. Engineering a more polar salt bridge between p51 and p66 resulted in even greater increases in the thumb/fingers opening, RT sliding, dNTP binding disruption and in vitro and in vivo RT inhibition than were observed with wild-type RT. We also observed that K103N, a clinically relevant NNRTI resistance mutation, does not prevent binding between efavirenz and RT-T/P but instead allows formation of a stable and productive RT-T/P-dNTP complex, possibly through disruption of the E138-K101 salt bridge. Collectively, these data describe unique structure-activity-resistance relationships that could be exploited for drug development.
Assuntos
Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/química , Inibidores da Transcriptase Reversa/farmacologia , Alcinos , Regulação Alostérica , Benzoxazinas/farmacologia , Ciclopropanos , Primers do DNA , Desoxirribonucleotídeos/metabolismo , Polarização de Fluorescência , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , Mutação , Subunidades Proteicas/química , Moldes GenéticosRESUMO
The essential DNA helicase, PcrA, regulates recombination by displacing the recombinase RecA from the DNA. The nucleotide-bound state of RecA determines the stability of its nucleoprotein filaments. Using single-molecule fluorescence approaches, we demonstrate that RecA displacement by a translocating PcrA requires the ATPase activity of the recombinase. We also show that in a 'head-on collision' between a polymerizing RecA filament and a translocating PcrA, the RecA K72R ATPase mutant, but not wild-type RecA, arrests helicase translocation. Our findings demonstrate that translocation of PcrA is not sufficient to displace RecA from the DNA and assigns an essential role for the ATPase activity of RecA in helicase-mediated disruption of its filaments.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , DNA Helicases/metabolismo , Recombinases Rec A/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , DNA de Cadeia Simples/metabolismo , Geobacillus stearothermophilus/enzimologia , Transporte ProteicoRESUMO
Replication stress describes various types of endogenous and exogenous challenges to DNA replication in S-phase. Stress during this critical process results in helicase-polymerase decoupling at replication forks, triggering the S-phase checkpoint, which orchestrates global replication fork stalling and delayed entry into G2. The replication stressor most often used to induce the checkpoint response is hydroxyurea (HU), a chemotherapeutic agent. The primary mechanism of S-phase checkpoint activation by HU has thus far been considered to be a reduction of dNTP synthesis by inhibition of ribonucleotide reductase (RNR), leading to helicase-polymerase decoupling and subsequent activation of the checkpoint, mediated by the replisome associated effector kinase Mrc1. In contrast, we observe that HU causes cell cycle arrest in budding yeast independent of both the Mrc1-mediated replication checkpoint response and the Psk1-Mrc1 oxidative signaling pathway. We demonstrate a direct relationship between HU incubation and reactive oxygen species (ROS) production in yeast nuclei. We further observe that ROS strongly inhibits the in vitro polymerase activity of replicative polymerases (Pols), Pol α, Pol δ, and Pol ε, causing polymerase complex dissociation and subsequent loss of DNA substrate binding, likely through oxidation of their integral iron sulfur Fe-S clusters. Finally, we present "RNR-deg," a genetically engineered alternative to HU in yeast with greatly increased specificity of RNR inhibition, allowing researchers to achieve fast, nontoxic, and more readily reversible checkpoint activation compared to HU, avoiding harmful ROS generation and associated downstream cellular effects that may confound interpretation of results.
RESUMO
The minichromosome maintenance (MCM) helicase complex is essential for the initiation and elongation of DNA replication in both the eukaryotic and archaeal domains. The archaeal homohexameric MCM helicase from Sulfolobus solfataricus serves as a model for understanding mechanisms of DNA unwinding. In this report, the displaced 5'-tail is shown to provide stability to the MCM complex on DNA and contribute to unwinding. Mutations in a positively charged patch on the exterior surface of the MCM hexamer destabilize this interaction, alter the path of the displaced 5'-tail DNA and reduce unwinding. DNA footprinting and single-molecule fluorescence experiments support a previously unrecognized wrapping of the 5'-tail. This mode of hexameric helicase DNA unwinding is termed the steric exclusion and wrapping (SEW) model, where the 3'-tail is encircled by the helicase while the displaced 5'-tail wraps around defined paths on the exterior of the helicase. The novel wrapping mechanism stabilizes the MCM complex in a positive unwinding mode, protects the displaced single-stranded DNA tail and prevents reannealing.
Assuntos
Proteínas Arqueais/química , DNA Helicases/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , DNA/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Mutação , Proteínas de Plantas/metabolismo , Ligação Proteica , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Sulfolobus solfataricus/enzimologiaRESUMO
R-loop proteins present a stable and robust blockade to the progression of a DNA replication fork during S-phase. The consequences of this block can include mutagenesis and other irreversible chromosomal catastrophes, causing genomic instability and disease. As such, further investigation into the molecular mechanisms underlying R-loop protein resolution is warranted. The critical role of non-replicative accessory helicases in R-loop protein resolution has increasingly come into light in recent years. Such helicases include the Pif1-family, monomeric helicases that have been studied in many different contexts and that have been ascribed to a multitude of separable protective functions in the cell. In this chapter, we present protocols to study R-loop protein resolution by Pif1 helicase at stalled replication forks using purified proteins, both at the biochemical and single-molecule level. Our system uses recombinant proteins expressed in Saccharomyces cerevisiae but could apply to practically any organism of interest due to the high interspecies homology of the proteins involved in DNA replication. The methods we outline are extensible to many systems and should be applicable to studying R-loop clearance by any Superfamily (SF) 1B helicase. These techniques will further enable mechanistic research on these critical but understudied components of the genomic maintenance program.
Assuntos
Estruturas R-Loop , Proteínas de Saccharomyces cerevisiae , DNA Helicases/química , Replicação do DNA , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The eukaryotic leading strand DNA polymerase (Pol) ε contains 4 subunits, Pol2, Dpb2, Dpb3 and Dpb4. Pol2 is a fusion of two B-family Pols; the N-terminal Pol module is catalytic and the C-terminal Pol module is non-catalytic. Despite extensive efforts, there is no atomic structure for Pol ε holoenzyme, critical to understanding how DNA synthesis is coordinated with unwinding and the DNA path through the CMG helicase-Pol ε-PCNA clamp. We show here a 3.5-Šcryo-EM structure of yeast Pol ε revealing that the Dpb3-Dpb4 subunits bridge the two DNA Pol modules of Pol2, holding them rigid. This information enabled an atomic model of the leading strand replisome. Interestingly, the model suggests that an OB fold in Dbp2 directs leading ssDNA from CMG to the Pol ε active site. These results complete the DNA path from entry of parental DNA into CMG to exit of daughter DNA from PCNA.
Assuntos
DNA Polimerase II/química , Replicação do DNA , Microscopia Crioeletrônica , Modelos Moleculares , Estrutura Molecular , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Limited experimental tools are available to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct as a generic, novel, targetable protein-DNA roadblock for studying mechanisms underlying enzymatic activities on DNA substrates in vitro. We illustrate the broad utility of this tool by demonstrating replication fork arrest by the specifically bound dCas9-guideRNA complex to arrest viral, bacterial and eukaryotic replication forks in vitro.
Assuntos
Proteína 9 Associada à CRISPR/genética , Replicação do DNA/genética , DNA Bacteriano/genética , Escherichia coli/genética , RNA Guia de Cinetoplastídeos/genética , Sistemas CRISPR-Cas/genética , Streptococcus pyogenes/enzimologiaRESUMO
The eukaryotic replisome is a multiprotein complex that duplicates DNA. The replisome is sculpted to couple continuous leading strand synthesis with discontinuous lagging strand synthesis, primarily carried out by DNA polymerases ε and δ, respectively, along with helicases, polymerase α-primase, DNA sliding clamps, clamp loaders and many other proteins. We have previously established the mechanisms by which the polymerases ε and δ are targeted to their 'correct' strands, as well as quality control mechanisms that evict polymerases when they associate with an 'incorrect' strand. Here, we provide a practical guide to differentially assay leading and lagging strand replication in vitro using pure proteins.
RESUMO
Replicative helicases in all cell types are hexameric rings that unwind DNA by steric exclusion in which the helicase encircles the tracking strand only and excludes the other strand from the ring. This mode of translocation allows helicases to bypass blocks on the strand that is excluded from the central channel. Unlike other replicative helicases, eukaryotic CMG helicase partially encircles duplex DNA at a forked junction and is stopped by a block on the non-tracking (lagging) strand. This report demonstrates that Mcm10, an essential replication protein unique to eukaryotes, binds CMG and greatly stimulates its helicase activity in vitro. Most significantly, Mcm10 enables CMG and the replisome to bypass blocks on the non-tracking DNA strand. We demonstrate that bypass occurs without displacement of the blocks and therefore Mcm10 must isomerize the CMG-DNA complex to achieve the bypass function.
Assuntos
DNA Helicases/metabolismo , Replicação do DNA , DNA Fúngico/metabolismo , Proteínas de Manutenção de Minicromossomo/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Complexos Multienzimáticos/metabolismoRESUMO
We have reconstituted a eukaryotic leading/lagging strand replisome comprising 31 distinct polypeptides. This study identifies a process unprecedented in bacterial replisomes. While bacteria and phage simply recruit polymerases to the fork, we find that suppression mechanisms are used to position the distinct eukaryotic polymerases on their respective strands. Hence, Pol ε is active with CMG on the leading strand, but it is unable to function on the lagging strand, even when Pol δ is not present. Conversely, Pol δ-PCNA is the only enzyme capable of extending Okazaki fragments in the presence of Pols ε and α. We have shown earlier that Pol δ-PCNA is suppressed on the leading strand with CMG (Georgescu et al., 2014). We propose that CMG, the 11-subunit helicase, is responsible for one or both of these suppression mechanisms that spatially control polymerase occupancy at the fork.
Assuntos
DNA Helicases/genética , Replicação do DNA , DNA Fúngico/genética , Subunidades Proteicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sequência de Bases , DNA/genética , DNA/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , DNA Polimerase I/genética , DNA Polimerase I/metabolismo , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , DNA Polimerase beta/genética , DNA Polimerase beta/metabolismo , DNA Fúngico/química , DNA Fúngico/metabolismo , Expressão Gênica , Dados de Sequência Molecular , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
BACKGROUND: A key challenge in interdisciplinary research is choosing the best approach from a large number of techniques derived from different disciplines and their interfaces. RESULTS: To address this challenge in the area of Biophysics and Structural Biology, we have designed a graduate level course to teach students insightful use of experimental biophysical approaches in relationship to addressing biological questions related to biomolecular interactions and dynamics. A weekly seminar and data and literature club are used to compliment the training in class. The course contains wet-laboratory experimental demonstration and real-data analysis as well as lectures, grant proposal preparation and assessment, and student presentation components. Active student participation is mandatory in all aspects of the class. Students prepare materials for the class receiving individual and iterative feedback from course directors and local experts generating high quality classroom presentations. CONCLUSIONS: The ultimate goal of the course is to teach students the skills needed to weigh different experimental approaches against each other in addressing a specific biological question by thinking and executing academic tasks like faculty.
RESUMO
HIV-1 reverse transcriptase (RT) plays a central role in HIV infection. Current United States Federal Drug Administration (USFDA)-approved antiretroviral therapies can include one of five approved non-nucleoside RT inhibitors (NNRTIs), which are potent inhibitors of RT activity. Despite their crucial clinical role in treating and preventing HIV-1 infection, their mechanism of action remains elusive. In this review, we introduce RT and highlight major advances from experimental and computational biophysical experiments toward an understanding of RT function and the inhibitory mechanism(s) of NNRTIs.