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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.
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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
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.
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DNA Helicases , Proteínas de Escherichia coli , DNA/metabolismo , DNA Helicases/química , Replicação do DNA , Escherichia coli/enzimologia , Proteínas de Escherichia coli/químicaRESUMO
Carbon-based hole transport layer-free perovskite solar cells (PSCs) based on methylammonium lead triiodide (MAPbI3 ) have become one of the research focus due to low cost, easy preparation, and good optoelectronic properties. However, instability of perovskite under vacancy defects and stress-strain makes it difficult to achieve high-efficiency and stable power output. Here, a soft-structured long-chain 2D pentanamine iodide (abbreviated as "PI") is used to improve perovskite quality and interfacial mechanical compatibility. PI containing CH3 (CH2 )4 NH3 + and I- ions not only passivate defects at grain boundaries, but also effectively alleviate residual stress during high temperature annealing via decreasing Young's modulus of perovskite film. Most importantly, PI effectively increases matching degree of Young's modulus between MAPbI3 (47.1 GPa) and carbon (6.7 GPa), and strengthens adhesive fracture energy (Gc ) between perovskite and carbon, which is helpful for outward release of nascent interfacial stress generated under service conditions. Consequently, photoelectric conversion efficiency (PCE) of optimal device is enhanced from 10.85% to 13.76% and operational stability is also significantly improved. 83.1% output is maintained after aging for 720 h at room temperature and 25-60% relative humidity (RH). This strategy of regulation from chemistry and physics provides a strategy for efficient and stable carbon-based PSCs.
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INTRODUCTION: Artemisia species are widely spread in north hemisphere. Artemisia sieversiana pollen is one of the common pollen allergens in the north of China. At present, seven allergens were identified and had been listed officially from A. sieversiana pollen, but the remaining allergens are still insufficiently studied, which need to be found. METHODS: Pectate lyase was purified from the extracts of A. sieversiana pollen by anion exchange, size exclusion, and HPLC-hydrophobic interaction chromatography. The gene of A. sieversiana pectate lyase (Art si pectate lyase) was cloned and expressed in Escherichia coli. The enzyme activity and circular dichroism (CD) spectrum of natural and recombinant proteins were analyzed. The allergenicity of Art si pectate lyase was characterized by enzyme-linked immunosorbent assay (ELISA), Western blot, inhibition ELISA, and basophil activation test. The allergen's physicochemical properties, three-dimensional structure, sequence profiles with homologous allergens and phylogenetic tree were analyzed by in silico methods. RESULTS: Natural Art si pectate lyase (nArt si pectate lyase) was purified from A. sieversiana pollen extracts by three chromatographic strategies. The cDNA sequence of Art si pectate lyase had a 1191-bp open reading frame encoding 396 amino acids. Both natural and recombinant pectate lyase (rArt si pectate lyase) exhibited similar CD spectrum, and nArt si pectate lyase had higher enzymatic activity. Moreover, the specific immunoglobulin E (IgE) binding rate against nArt si pectate lyase and rArt si pectate lyase was determined as 40% (6/15) in patients' serum with Artemisia species pollen allergy by ELISA. The nArt si pectate lyase and rArt si pectate lyase could inhibit 76.11% and 47.26% of IgE binding activities to the pollen extracts, respectively. Art si pectate lyase was also confirmed to activate patients' basophils. Its structure contains a predominant motif of classic parallel helical core, consisting of three parallel ß-sheets, and two highly conserved features (vWiDH, RxPxxR) which may contribute to pectate lyase activity. Moreover, Art si pectate lyase shared the highest sequence identity of 73.0% with Art v 6 among currently recognized pectate lyase allergen, both were clustered into the same branch in the phylogenetic tree. CONCLUSION: In this study, pectate lyase was identified and comprehensively characterized as a novel allergen in A. sieversiana pollen. The findings enriched the allergen information for this pollen and promoted the development of component-resolved diagnosis and molecular therapy of A. sieversiana pollen allergy.
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INTRODUCTION: Recombinant allergens produced by Escherichia coli (E. coli) system play an important role in the component-resolved diagnostics of allergy and vaccine development. However, incorrect folding of recombinant allergens may affect their application. Therefore, it is very important to monitor the correct folding of recombinant allergens. Currently, there is still a lack of a quality control strategy to solve this problem. In this study, a mite allergen, Der f 2, was taken as an example to establish a novel quality control strategy, which was based on chromatography to isolate the allergen, and on enzyme-linked immunosorbent assay to verify the IgE reactivity of the isolated allergen. METHODS: The nucleotide sequence encoding Der f 2 was codon-optimized and cloned into pET-28a (+) plasmid. Best conditions for the expression of Der f 2 in E. coli were sought. The inclusion body of Der f 2 was denatured and purified by nickel affinity chromatography. Refolding processes were compared using glutathione redox system. The fully and partially folded proteins were separated by anion exchange chromatography, and the IgE reactivity of the isolated proteins was verified by indirect enzyme-linked immunosorbent assay. RESULTS: An optimized 387 bp segment of the Der f 2 coding gene was successfully expressed in E. coli. Best induction conditions included preinduction bacterial density with absorbance value at 600 nm was 0.6, 1 mM isopropyl beta-
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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
BACKGROUND: This study aimed to assess the radiological and clinical outcomes of treatment using the ankle dislocation method for posterior malleolar malunion. METHOD: Thirty-one patients with posterior malleolar malunion who underwent treatment using the ankle dislocation method from May 2015 to October 2021 were retrospectively analyzed. Key outcome measures were radiographic parameters (articular step-off, tibiofibular clear space, fibular length, tibial lateral surface angle, and ankle osteoarthritis), clinical scores (American Orthopaedic Foot and Ankle Society ankle-hindfoot scale and Visual Analogue Scale), and patient satisfaction rate. RESULT: Preoperative computed tomography revealed that Bartoní cek types 3 and 4 accounted for 64.5 % (n = 20) of total cases. Most posterior malleolar malunions were accompanied by depressed intercalary fragments (61.2 % [n = 19]). At the final follow-up, radiographic parameters and clinical scores showed significant improvements postoperatively (P < 0.05), with a high patient satisfaction rate of 77.4 %. Subgroup analysis revealed that the posterior malleolar fracture morphology significantly affected postoperative pain, particularly in more complex fractures (P < 0.001). CONCLUSION: The ankle dislocation method effectively exposes the distal tibial articular surface and facilitates the anatomical restoration of joint congruity under direct vision. This approach substantially improves the clinical and imaging outcomes in patients with complex posterior malleolar malunion. LEVELS OF EVIDENCE: Level IV, retrospective case series.
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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.
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Replicação do DNA , DnaB Helicases/metabolismo , DNA Polimerase Dirigida por DNA , Escherichia coli/genética , Complexos Multienzimáticos , Imagem Individual de MoléculaRESUMO
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.
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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.
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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
Neurofibrillary tangles of hyperphosphorylated tau protein (p-tau) are a key pathological feature of Alzheimer's disease (AD). Tau phosphorylation is suggested to be secondary to amyloid-beta (Aß) accumulation. However, the mechanism by which Aß induces tau phosphorylation in neurons remains unclear. Neurotrophin receptor p75 (p75NTR) is a receptor for Aß and mediates Aß neurotoxicity, implying that p75NTR may mediate Aß-induced tau phosphorylation in AD. Here, we showed that Aß-induced tau hyperphosphorylation and neurodegeneration, including tau phosphorylation, synaptic disorder and neuronal loss, in the brains of both male wild-type (Wt) mice and male P301L transgenic mice (a mouse model of human tauopathy) were alleviated by genetic knockout of p75NTR in the both mouse models. We further confirmed that the activation or inhibition of cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase-3ß (GSK3ß) significantly changed Aß/p75NTR-mediated p-tau levels in neurons. Treatment of male P301L mice with soluble p75NTR extracellular domain (p75ECD-Fc), which antagonizes the binding of Aß to p75NTR, suppressed tau hyperphosphorylation. Taken together, our findings suggest that p75NTR meditates Aß-induced tau pathology and is a potential druggable target for AD and other tauopathies.
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Peptídeos beta-Amiloides/toxicidade , Receptores de Fator de Crescimento Neural/metabolismo , Tauopatias/metabolismo , Proteínas tau/metabolismo , Animais , Células Cultivadas , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Distribuição Aleatória , Receptores de Fator de Crescimento Neural/administração & dosagem , Receptores de Fator de Crescimento Neural/genética , Tauopatias/tratamento farmacológico , Tauopatias/genética , Proteínas tau/antagonistas & inibidores , Proteínas tau/genéticaRESUMO
Tau pathology is characterized as a form of frontotemporal lobar degeneration (FTLD) known as FTLD-tau. The underlying pathogenic mechanisms are not known and no therapeutic interventions are currently available. Here, we report that the neurotrophin receptor p75NTR plays a critical role in the pathogenesis of FTLD-tau. The expression of p75NTR and the precursor of nerve growth factor (proNGF) were increased in the brains of FTLD-tau patients and mice (P301L transgenic). ProNGF-induced tau phosphorylation via p75NTR in vitro, which was associated with the AKT/glycogen synthase kinase (GSK)3ß pathway. Genetic reduction of p75NTR in P301L mice rescued the memory deficits, alleviated tau hyperphosphorylation and restored the activity of the AKT/GSK3ß pathway. Treatment of the P301L mice with the soluble p75NTR extracellular domain (p75ECD-Fc), which can antagonize neurotoxic ligands of p75NTR, effectively improved memory behavior and suppressed tau pathology. This suggests that p75NTR plays a crucial role in tau paGSKthology and represents a potential druggable target for FTLD-tau and related tauopathies.
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Degeneração Lobar Frontotemporal/metabolismo , Fator de Crescimento Neural/metabolismo , Precursores de Proteínas/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Proteínas tau/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Células Cultivadas , Feminino , Degeneração Lobar Frontotemporal/patologia , Degeneração Lobar Frontotemporal/terapia , Glicogênio Sintase Quinase 3 beta/metabolismo , Masculino , Transtornos da Memória/metabolismo , Transtornos da Memória/patologia , Transtornos da Memória/terapia , Camundongos Transgênicos , Neurônios/metabolismo , Neurônios/patologia , Fosforilação/fisiologia , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de SinaisRESUMO
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.
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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
BACKGROUND: To investigate the effects of abdominal obesity (AO) and nonalcoholic fatty liver disease (NAFLD) with or without AO on carotid arteries by determining carotid intima-media thickness (CIMT). METHODS: A total of 2745 Chinese Han adults (aged between 40 and 50 years old) were recruited and divided into 4 groups: (1) NW-no NAFL group: the normal body weight without NAFLD (n = 1888); (2) AO-no NAFL group: AO without NAFLD (n = 259); (3) NW-with NAFL group: NAFLD without AO (n = 93); and (4) AO-with NAFL group: AO with NAFLD (n = 505). The CIMT rate of each group was compared among 4 groups and the regression analysis was further used to correct confounders. RESULTS: We found that the NW-with NAFL group had a significantly higher CIMT rate than the AO-no NAFL group ([.87 ± .31] versus [.72 ± .29] P < .01) and the AO-with NAFL group ([.87 ± .31] versus [.79 ± .26], P < .01). CONCLUSIONS: The ectopic liver fat accumulation may increase the risk of atherosclerosis. Therefore, screening NAFLD in the population with normal weight may be beneficial for the prevention of atherosclerosis at an early stage.
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Artérias Carótidas/diagnóstico por imagem , Espessura Intima-Media Carotídea , Hepatopatia Gordurosa não Alcoólica/diagnóstico por imagem , Obesidade Abdominal/diagnóstico por imagem , Tecido Adiposo/diagnóstico por imagem , Adulto , Aterosclerose/diagnóstico por imagem , Composição Corporal , China , Feminino , Humanos , Fígado/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Análise de Regressão , Fatores de RiscoRESUMO
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.
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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
To understand the status of food-borne parasitic infection in intermediate hosts in Ninghai County of Zhejiang Province, freshwater crabs were collected from 4 towns of the County to detect the infection with metacercariae of Paragonimus, and frogs were collected from 8 towns to examine the infection with plerocercoid of Spirometra mansoni from May to September, 2015. Among the 339 freshwater crabs collected, the infection rate was 9.1%ï¼31/339ï¼, with each crab containing 6.7 metacercariae of Paragonimus on average. Among the 348 frogs collected, the infection rate was 11.5%ï¼40/348ï¼, with each frog containing 2.2 plerocercoids on average. In conclusion, there is a high rate of food-borne parasitic infection in intermediate hosts in Ninghai County. Comprehensive prevention and control measures are needed.
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Braquiúros/parasitologia , Animais , Doenças Transmissíveis , Alimentos , Água Doce , MetacercáriasRESUMO
We demonstrate sensitive detection of alpha protons of fully protonated proteins by solid-state NMR spectroscopy with 100-111â kHz magic-angle spinning (MAS). The excellent resolution in the Cα-Hα plane is demonstrated for 5â proteins, including microcrystals, a sedimented complex, a capsid and amyloid fibrils. A set of 3D spectra based on a Cα-Hα detection block was developed and applied for the sequence-specific backbone and aliphatic side-chain resonance assignment using only 500â µg of sample. These developments accelerate structural studies of biomolecular assemblies available in submilligram quantities without the need of protein deuteration.
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Amiloide/química , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Acinetobacter/virologia , Animais , Bacteriófagos/química , Cristalização , Humanos , Nucleocapsídeo/química , Multimerização Proteica , PrótonsRESUMO
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.