RESUMO
The simian virus 40 (SV40) replisome only encodes for its helicase; large T-antigen (L-Tag), while relying on the host for the remaining proteins, making it an intriguing model system. Despite being one of the earliest reconstituted eukaryotic systems, the interactions coordinating its activities and the identification of new factors remain largely unexplored. Herein, we in vitro reconstituted the SV40 replisome activities at the single-molecule level, including DNA unwinding by L-Tag and the single-stranded DNA-binding protein Replication Protein A (RPA), primer extension by DNA polymerase δ, and their concerted leading-strand synthesis. We show that RPA stimulates the processivity of L-Tag without altering its rate and that DNA polymerase δ forms a stable complex with L-Tag during leading-strand synthesis. Furthermore, similar to human and budding yeast Cdc45-MCM-GINS helicase, L-Tag uses the fork protection complex (FPC) and the mini-chromosome maintenance protein 10 (Mcm10) during synthesis. Hereby, we demonstrate that FPC increases this rate, and both FPC and Mcm10 increase the processivity by stabilizing stalled replisomes and increasing their chances of restarting synthesis. The detailed kinetics and novel factors of the SV40 replisome establish it as a closer mimic of the host replisome and expand its application as a model replication system.
Assuntos
Replicação do DNA , Proteínas de Manutenção de Minicromossomo , Proteína de Replicação A , Vírus 40 dos Símios , Vírus 40 dos Símios/metabolismo , Vírus 40 dos Símios/genética , Humanos , Proteínas de Manutenção de Minicromossomo/metabolismo , Proteínas de Manutenção de Minicromossomo/genética , Proteína de Replicação A/metabolismo , DNA Polimerase III/metabolismo , DNA Polimerase III/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , DNA Helicases/metabolismo , DNA Helicases/genética , DNA Viral/metabolismo , DNA Viral/genética , Replicação Viral , Imagem Individual de Molécula , Antígenos Transformantes de Poliomavirus/metabolismo , Antígenos Transformantes de Poliomavirus/genética , DNA de Cadeia Simples/metabolismo , DNA Polimerase Dirigida por DNA , Complexos MultienzimáticosRESUMO
Numerous eukaryotic DNA processing enzymes, such as DNA polymerases and ligases, bind the processivity factor PCNA, which acts as a platform to recruit and regulate the binding of enzymes to their DNA substrate. Multiple PCNA-interacting motifs (PIPs) are present in these enzymes, but their individual structural and functional role has been a matter of debate. Recent cryo-EM reconstructions of high-fidelity DNA polymerase Pol δ (Pol δ), translesion synthesis DNA polymerase κ (Pol κ) and Ligase 1 (Lig1) bound to a DNA substrate and PCNA demonstrate that the critical interaction with PCNA involves the internal PIP proximal to the catalytic domain. The ancillary PIPs, located in long disordered regions, are instead invisible in the reconstructions, and appear to function as flexible tethers when the enzymes fall off the DNA. In this review, we discuss the recent structural advancements and propose a functional hierarchy for the PIPs in Pol δ, Pol κ, and Lig1.
Assuntos
DNA Polimerase Dirigida por DNA , DNA , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , DNA Polimerase Dirigida por DNA/metabolismo , DNA/genética , Replicação do DNA , DNA Polimerase III/química , DNA Polimerase III/genética , DNA Polimerase III/metabolismoRESUMO
The activity of enzymes is traditionally characterised through bulk-phase biochemical methods that only report on population averages. Single-molecule methods are advantageous in elucidating kinetic and population heterogeneity but are often complicated, time consuming, and lack statistical power. We present a highly-generalisable and high-throughput single-molecule assay to rapidly characterise proteins involved in DNA metabolism. The assay exclusively relies on changes in total fluorescence intensity of surface-immobilised DNA templates as a result of DNA synthesis, unwinding or digestion. Combined with an automated data-analysis pipeline, our method provides enzymatic activity data of thousands of molecules in less than an hour. We demonstrate our method by characterising three fundamentally different enzyme activities: digestion by the phage λ exonuclease, synthesis by the phage Phi29 polymerase, and unwinding by the E. coli UvrD helicase. We observe the previously unknown activity of the UvrD helicase to remove neutravidin bound to 5'-, but not 3'-ends of biotinylated DNA.
Assuntos
DNA Helicases , DNA , DNA/metabolismo , DNA Helicases/metabolismo , DNA de Cadeia Simples , Escherichia coli , Proteínas de Escherichia coli/metabolismo , CinéticaRESUMO
Nucleotide excision repair (NER) is critical for removing bulky DNA base lesions and avoiding diseases. NER couples lesion recognition by XPC to strand separation by XPB and XPD ATPases, followed by lesion excision by XPF and XPG nucleases. Here, we describe key regulatory mechanisms and roles of XPG for and beyond its cleavage activity. Strikingly, by combing single-molecule imaging and bulk cleavage assays, we found that XPG binding to the 7-subunit TFIIH core (coreTFIIH) stimulates coreTFIIH-dependent double-strand (ds)DNA unwinding 10-fold, and XPG-dependent DNA cleavage by up to 700-fold. Simultaneous monitoring of rates for coreTFIIH single-stranded (ss)DNA translocation and dsDNA unwinding showed XPG acts by switching ssDNA translocation to dsDNA unwinding as a likely committed step. Pertinent to the NER pathway regulation, XPG incision activity is suppressed during coreTFIIH translocation on DNA but is licensed when coreTFIIH stalls at the lesion or when ATP hydrolysis is blocked. Moreover, ≥15 nucleotides of 5'-ssDNA is a prerequisite for efficient translocation and incision. Our results unveil a paired coordination mechanism in which key lesion scanning and DNA incision steps are sequentially coordinated, and damaged patch removal is only licensed after generation of ≥15 nucleotides of 5'-ssDNA, ensuring the correct ssDNA bubble size before cleavage.
Nucleotide excision repair (NER) removes bulky DNA lesions and is thereby crucial in maintaining transcription and genomic integrity. Here, the authors show a dual function for the XPG nuclease that is critical for finding and excising the damage. During the separation of the damage-containing strand from the undamaged strand, XPG stimulates TFIIH dependent dsDNA unwinding 10 fold. In return, when TFIIH stalls at the damage it stimulates XPG nuclease activity 700 fold. Remarkably, this mutually exclusive coordination requires a bubble longer than 15 nucleotides. This study addressees why a bubble of a certain size is needed to facilitate NER and why XPG is recruited at the beginning of NER when its endonucleolytic activity is required at the very end.
Assuntos
Reparo do DNA , Fator de Transcrição TFIIH , DNA/metabolismo , Dano ao DNA , DNA de Cadeia Simples , Endonucleases/metabolismo , Nucleotídeos , Fator de Transcrição TFIIH/metabolismoRESUMO
DNA strand breaks are repaired by DNA synthesis from an exposed DNA end paired with a homologous DNA template. DNA polymerase delta (Pol δ) catalyses DNA synthesis in multiple eukaryotic DNA break repair pathways but triggers genome instability unless its activity is restrained. We show that human HelQ halts DNA synthesis by isolated Pol δ and Pol δ-PCNA-RPA holoenzyme. Using novel HelQ mutant proteins we identify that inhibition of Pol δ is independent of DNA binding, and maps to a 70 amino acid intrinsically disordered region of HelQ. Pol δ and its POLD3 subunit robustly stimulated DNA single-strand annealing by HelQ, and POLD3 and HelQ interact physically via the intrinsically disordered HelQ region. This data, and inability of HelQ to inhibit DNA synthesis by the POLD1 catalytic subunit of Pol δ, reveal a mechanism for limiting DNA synthesis and promoting DNA strand annealing during human DNA break repair, which centres on POLD3.
Assuntos
DNA Helicases , DNA Polimerase III , Replicação do DNA , Humanos , DNA/metabolismo , DNA Polimerase III/genética , Primers do DNA , Antígeno Nuclear de Célula em Proliferação/metabolismo , DNA Helicases/química , DNA Helicases/metabolismoRESUMO
Replisomes are the protein assemblies that replicate DNA. They function as molecular motors to catalyze template-mediated polymerization of nucleotides, unwinding of DNA, the synthesis of RNA primers, and the assembly of proteins on DNA. The replisome of bacteriophage T7 contains a minimum of proteins, thus facilitating its study. This review describes the molecular motors and coordination of their activities, with emphasis on the T7 replisome. Nucleotide selection, movement of the polymerase, binding of the processivity factor, unwinding of DNA, and RNA primer synthesis all require conformational changes and protein contacts. Lagging-strand synthesis is mediated via a replication loop whose formation and resolution is dictated by switches to yield Okazaki fragments of discrete size. Both strands are synthesized at identical rates, controlled by a molecular brake that halts leading-strand synthesis during primer synthesis. The helicase serves as a reservoir for polymerases that can initiate DNA synthesis at the replication fork. We comment on the differences in other systems where applicable.
Assuntos
Bacteriófago T7/metabolismo , Replicação do DNA , Bacteriófago T4/genética , Bacteriófago T4/metabolismo , Bacteriófago T7/química , Bacteriófago T7/genética , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/virologiaRESUMO
A cell's ability to survive and to evade cancer is contingent on its ability to retain genomic integrity, which can be seriously compromised when nucleic acid phosphodiester bonds are disrupted. DNA Ligase 1 (LIG1) plays a key role in genome maintenance by sealing single-stranded nicks that are produced during DNA replication and repair. Autosomal recessive mutations in a limited number of individuals have been previously described for this gene. Here we report a homozygous LIG1 mutation (p.A624T), affecting a universally conserved residue, in a patient presenting with leukopenia, neutropenia, lymphopenia, pan-hypogammaglobulinemia, and diminished in vitro response to mitogen stimulation. Patient fibroblasts expressed normal levels of LIG1 protein but exhibited impaired growth, poor viability, high baseline levels of gamma-H2AX foci, and an enhanced susceptibility to DNA-damaging agents. The mutation reduced LIG1 activity by lowering its affinity for magnesium 2.5-fold. Remarkably, it also increased LIG1 fidelity > 50-fold against 3' end 8-Oxoguanine mismatches, exhibiting a marked reduction in its ability to process such nicks. This is expected to yield increased ss- and dsDNA breaks. Molecular dynamic simulations, and Residue Interaction Network studies, predicted an allosteric effect for this mutation on the protein loops associated with the LIG1 high-fidelity magnesium, as well as on DNA binding within the adenylation domain. These dual alterations of suppressed activity and enhanced fidelity, arising from a single mutation, underscore the mechanistic picture of how a LIG1 defect can lead to severe immunological disease.
Assuntos
DNA Ligase Dependente de ATP , Homozigoto , Mutação , Imunodeficiência Combinada Severa , Feminino , Humanos , Masculino , DNA Ligase Dependente de ATP/genética , DNA Ligase Dependente de ATP/metabolismo , Fibroblastos , Simulação de Dinâmica Molecular , Mutação/genética , Imunodeficiência Combinada Severa/genética , LactenteRESUMO
Dicer-like 1 (DCL1) is a core component of the plant microRNA (miRNA) biogenesis machinery. MiRNA is transcribed as a precursor RNA, termed primary miRNA (pri-miRNA), which is cleaved by DCL1 in two steps to generate miRNA/miRNA* duplex. Pri-miRNA is a single-stranded RNA that forms a hairpin structure with a number of unpaired bases, hereafter called mismatches, on its stem. In the present study, by using purified recombinant Arabidopsis DCL1, we presented evidence that mismatches on the stem of pri-miRNA are important for precise DCL1 cleavage. We showed that a mismatch at the loop-distal side of the end of miRNA/miRNA* duplex is important for efficient cleavage of pri-miRNA in vitro, as previously suggested in planta. On the contrary, mismatches distant from the miRNA/miRNA* duplex region are important for determining the cleavage position by DCL1. The purified DCL1 proteins cleaved mutant pri-miRNA variants without such mismatches at a position at which wild-type pri-miRNA variants are not usually cleaved, resulting in an increased accumulation of small RNA different from miRNA. Therefore, our results suggest that, in addition to the distance from the ssRNA-dsRNA junction, mismatches on the stem of pri-miRNA function as a determinant for precise processing of pri-miRNA by DCL1 in plants.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Ciclo Celular , MicroRNAs , Ribonuclease III , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Nucleotídeos/metabolismo , Processamento Pós-Transcricional do RNA , Ribonuclease III/genética , Ribonuclease III/metabolismoRESUMO
The polyhistidine tag (His-tag) is one of the most popular protein tags used in the life sciences. Traditionally, the detection of His-tagged proteins relies on immunoblotting with anti-His antibodies. This approach is laborious for certain applications, such as protein purification, where time and simplicity are critical. The His-tag can also be directly detected by metal ion-loaded nickel-nitrilotriacetic acid-based chelator heads conjugated to fluorophores, which is a convenient alternative method to immunoblotting. Typically, such chelator heads are conjugated to either green or red fluorophores, the detection of which requires specialized excitation sources and detection systems. Here, we demonstrate that post-run staining is ideal for His-tag detection by metal ion-loaded and fluorescently labeled chelator heads in PAGE and blot membranes. Additionally, by comparing the performances of different chelator heads, we show how differences in microscopic affinity constants translate to macroscopic differences in the detection limits in environments with limited diffusion, such as PAGE. On the basis of these results, we devise a simple approach, called UVHis-PAGE, that uses metal ion-loaded and fluorescently labeled chelator heads to detect His-tagged proteins in PAGE and blot membranes. Our method uses a UV transilluminator as an excitation source, and the results can be visually inspected by the naked eye.
Assuntos
Eletroforese em Gel de Gradiente Desnaturante , Corantes Fluorescentes/química , Histidina/análise , Proteínas Recombinantes de Fusão/análise , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/análise , Raios Ultravioleta , Histidina/química , Humanos , Proteínas Recombinantes de Fusão/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genéticaRESUMO
Selectins are key to mediating interactions involved in cellular adhesion and migration, underlying processes such as immune responses, metastasis, and transplantation. Selectins are composed of a lectin domain, an epidermal growth factor (EGF)-like domain, multiple short consensus repeats (SCRs), a transmembrane domain, and a cytoplasmic tail. It is well-established that the lectin and EGF domains are required to mediate interactions with ligands; however, the contributions of the other domains in mediating these interactions remain obscure. Using various E-selectin constructs produced in a newly developed silkworm-based expression system and several assays performed under both static and physiological flow conditions, including flow cytometry, glycan array analysis, surface plasmon resonance, and cell-rolling assays, we show here that a reduction in the number of SCR domains is correlated with a decline in functional E-selectin binding to hematopoietic cell E- and/or L-selectin ligand (HCELL) and P-selectin glycoprotein ligand-1 (PSGL-1). Moreover, the binding was significantly improved through E-selectin dimerization and by a substitution (A28H) that mimics an extended conformation of the lectin and EGF domains. Analyses of the association and dissociation rates indicated that the SCR domains, conformational extension, and dimerization collectively contribute to the association rate of E-selectin-ligand binding, whereas just the lectin and EGF domains contribute to the dissociation rate. These findings provide the first evidence of the critical role of the association rate in functional E-selectin-ligand interactions, and they highlight that the SCR domains have an important role that goes beyond the structural extension of the lectin and EGF domains.
Assuntos
Selectina E/química , Selectina E/metabolismo , Animais , Bombyx , Linhagem Celular Tumoral , Selectina E/isolamento & purificação , Humanos , Proteínas Imobilizadas/metabolismo , Cinética , Ligantes , Camundongos , Polissacarídeos/metabolismo , Domínios Proteicos , Multimerização Proteica , Relação Estrutura-AtividadeRESUMO
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
Real-time visualization of single-proteins or -complexes on nucleic acid substrates is an essential tool for characterizing nucleic acid binding proteins. Here, we present a novel surface-condition independent and high-throughput single-molecule optical imaging platform called 'DNA skybridge'. The DNA skybridge is constructed in a 3D structure with 4 µm-high thin quartz barriers in a quartz slide. Each DNA end is attached to the top of the adjacent barrier, resulting in the extension and immobilization of DNA. In this 3D structure, the bottom surface is out-of-focus when the target molecules on the DNA are imaged. Moreover, the DNA skybridge itself creates a thin Gaussian light sheet beam parallel to the immobilized DNA. This dual property allows for imaging a single probe-tagged molecule moving on DNA while effectively suppressing interference with the surface and background signals from the surface.
Assuntos
DNA/ultraestrutura , Ensaios de Triagem em Larga Escala/métodos , Ácidos Nucleicos Imobilizados/ultraestrutura , Imagem Individual de Molécula/métodos , Nanotecnologia/métodos , Imagem Óptica/métodosRESUMO
Human GEN1 is a cytosolic homologous recombination protein that resolves persisting four-way Holliday junctions (HJ) after the dissolution of the nuclear membrane. GEN1 dimerization has been suggested to play key role in the resolution of the HJ, but the kinetic details of its reaction remained elusive. Here, single-molecule FRET shows how human GEN1 binds the HJ and always ensures its resolution within the lifetime of the GEN1-HJ complex. GEN1 monomer generally follows the isomer bias of the HJ in its initial binding and subsequently distorts it for catalysis. GEN1 monomer remains tightly bound with no apparent dissociation until GEN1 dimer is formed and the HJ is fully resolved. Fast on- and slow off-rates of GEN1 dimer and its increased affinity to the singly-cleaved HJ enforce the forward reaction. Furthermore, GEN1 monomer binds singly-cleaved HJ tighter than intact HJ providing a fail-safe mechanism if GEN1 dimer or one of its monomers dissociates after the first cleavage. The tight binding of GEN1 monomer to intact- and singly-cleaved HJ empowers it as the last resort to process HJs that escape the primary mechanisms.
Assuntos
DNA Cruciforme/genética , Resolvases de Junção Holliday/genética , Recombinação Genética , Dimerização , Endodesoxirribonucleases/genética , Recombinação Homóloga/genética , Humanos , Membrana Nuclear/genéticaRESUMO
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
RNA-DNA hybrid primers synthesized by low fidelity DNA polymerase α to initiate eukaryotic lagging strand synthesis must be removed efficiently during Okazaki fragment (OF) maturation to complete DNA replication. In this process, each OF primer is displaced and the resulting 5'-single-stranded flap is cleaved by structure-specific 5'-nucleases, mainly Flap Endonuclease 1 (FEN1), to generate a ligatable nick. At least two models have been proposed to describe primer removal, namely short- and long-flap pathways that involve FEN1 or FEN1 along with Replication Protein A (RPA) and Dna2 helicase/nuclease, respectively. We addressed the question of pathway choice by studying the kinetic mechanism of FEN1 action on short- and long-flap DNA substrates. Using single molecule FRET and rapid quench-flow bulk cleavage assays, we showed that unlike short-flap substrates, which are bound, bent and cleaved within the first encounter between FEN1 and DNA, long-flap substrates can escape cleavage even after DNA binding and bending. Notably, FEN1 can access both substrates in the presence of RPA, but bending and cleavage of long-flap DNA is specifically inhibited. We propose that FEN1 attempts to process both short and long flaps, but occasional missed cleavage of the latter allows RPA binding and triggers the long-flap OF maturation pathway.
Assuntos
Acetiltransferases/genética , Clivagem do DNA , Replicação do DNA/genética , DNA/genética , Proteínas de Membrana/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Acetiltransferases/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Cinética , Proteínas de Membrana/metabolismo , Ligação Proteica , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/genética , Imagem Individual de Molécula/métodos , Especificidade por SubstratoRESUMO
Flap endonucleases catalyze cleavage of single-stranded DNA flaps formed during replication, repair, and recombination and are therefore essential for genome processing and stability. Recent crystal structures of DNA-bound human flap endonuclease (hFEN1) offer new insights into how conformational changes in the DNA and hFEN1 may facilitate the reaction mechanism. For example, previous biochemical studies of DNA conformation performed under non-catalytic conditions with Ca2+ have suggested that base unpairing at the 5'-flap:template junction is an important step in the reaction, but the new structural data suggest otherwise. To clarify the role of DNA changes in the kinetic mechanism, we measured a series of transient steps, from substrate binding to product release, during the hFEN1-catalyzed reaction in the presence of Mg2+ We found that whereas hFEN1 binds and bends DNA at a fast, diffusion-limited rate, much slower Mg2+-dependent conformational changes in DNA around the active site are subsequently necessary and rate-limiting for 5'-flap cleavage. These changes are reported overall by fluorescence of 2-aminopurine at the 5'-flap:template junction, indicating that local DNA distortion (e.g. disruption of base stacking observed in structures), associated with positioning the 5'-flap scissile phosphodiester bond in the hFEN1 active site, controls catalysis. hFEN1 residues with distinct roles in the catalytic mechanism, including those binding metal ions (Asp-34 and Asp-181), steering the 5'-flap through the active site and binding the scissile phosphate (Lys-93 and Arg-100), and stacking against the base 5' to the scissile phosphate (Tyr-40), all contribute to these rate-limiting conformational changes, ensuring efficient and specific cleavage of 5'-flaps.
Assuntos
Cálcio/química , DNA/química , Endonucleases Flap/química , Magnésio/química , Cálcio/metabolismo , Catálise , Domínio Catalítico , DNA/metabolismo , Endonucleases Flap/metabolismo , Humanos , Magnésio/metabolismoRESUMO
The deep-sea brines of the Red Sea are remote and unexplored environments characterized by high temperatures, anoxic water, and elevated concentrations of salt and heavy metals. This environment provides a rare system to study the interplay between halophilic and thermophilic adaptation in biologic macromolecules. The present article reports the first DNA polymerase with halophilic and thermophilic features. Biochemical and structural analysis by Raman and circular dichroism spectroscopy showed that the charge distribution on the protein's surface mediates the structural balance between stability for thermal adaptation and flexibility for counteracting the salt-induced rigid and nonfunctional hydrophobic packing. Salt bridge interactions via increased negative and positive charges contribute to structural stability. Salt tolerance, conversely, is mediated by a dynamic structure that becomes more fixed and functional with increasing salt concentration. We propose that repulsive forces among excess negative charges, in addition to a high percentage of negatively charged random coils, mediate this structural dynamism. This knowledge enabled us to engineer a halophilic version of Thermococcus kodakarensis DNA polymerase.-Takahashi, M., Takahashi, E., Joudeh, L. I., Marini, M., Das, G., Elshenawy, M. M., Akal, A., Sakashita, K., Alam, I., Tehseen, M., Sobhy, M. A., Stingl, U., Merzaban, J. S., Di Fabrizio, E., Hamdan, S. M. Dynamic structure mediates halophilic adaptation of a DNA polymerase from the deep-sea brines of the Red Sea.
Assuntos
Proteínas Arqueais/química , DNA Polimerase Dirigida por DNA/química , Simulação de Dinâmica Molecular , Thermococcus/enzimologia , Oceano ÍndicoRESUMO
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 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
Selectins (E-, P-, and L-selectins) interact with glycoprotein ligands to mediate the essential tethering/rolling step in cell transport and delivery that captures migrating cells from the circulating flow. In this work, we developed a real time immunoprecipitation assay on a surface plasmon resonance chip that captures native glycoforms of two well known E-selectin ligands (CD44/hematopoietic cell E-/L-selectin ligand and P-selectin glycoprotein ligand-1) from hematopoietic cell extracts. Here we present a comprehensive characterization of their binding to E-selectin. We show that both ligands bind recombinant monomeric E-selectin transiently with fast on- and fast off-rates, whereas they bind dimeric E-selectin with remarkably slow on- and off-rates. This binding requires the sialyl Lewis x sugar moiety to be placed on both O- and N-glycans, and its association, but not dissociation, is sensitive to the salt concentration. Our results suggest a mechanism through which monomeric selectins mediate initial fast on and fast off kinetics to help capture cells out of the circulating shear flow; subsequently, tight binding by dimeric/oligomeric selectins is enabled to significantly slow rolling.