Jon R Sayers

The University of Sheffield | Sheffield · Department of Infection, Immunity & Cardiovascular Disease

Little is known about SARS-CoV-2 infection risk in African countries with high levels of infection-driven immunity and low vaccine coverage. We conducted a prospective cohort study of 349 participants from 52 households in The Gambia between March 2021 and June 2022, with routine weekly SARS-CoV-2 RT-PCR and 6-monthly SARS-CoV-2 serology. Attack ra...

The fundamental biology of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) nucleocapsid protein (Ncap), its use in diagnostic assays and its potential application as a vaccine component have received considerable attention since the outbreak of the Covid19 pandemic in late 2019. Here we report the scalable expression and purification o...

Flap endonucleases (FENs) recognise and cleave DNA substrates containing a 5'-single-strand (ss) of nucleic acid branching off a double-stranded (ds) DNA to yield a nicked duplex during DNA replication. Dynamic Atomic Force Microscopy of an inactive FEN mutant complexed with branched DNA revealed mobilisation of immobilised DNA, indicating that pro...

The fundamental biology of Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (Ncap), its use in diagnostic assays and its potential application as a vaccine component have received considerable attention since the outbreak of the Covid19 pandemic in late 2019. Here we report the scalable expression and purification o...

The prevalence and strength of serological responses mounted toward SARS-CoV-2 proteins other than nucleocapsid (N) and spike (S), which may be of use as additional serological markers, remains underexplored. Using high-content microscopy to assess antibody responses against full-length StrepTagged SARS-CoV-2 proteins, we found that 85% (166/196) o...

Bacterial pathogens are confronted with a range of challenges at the site of infection, including exposure to antibiotic treatment and harsh physiological conditions, that can alter the fitness benefits and costs of acquiring antibiotic resistance. Here, we develop an experimental system to recapitulate resistance gene acquisition by Staphylococcus...

Background: We aimed to measure SARS-CoV-2 seroprevalence in a cohort of healthcare workers (HCWs) during the first UK wave of the COVID-19 pandemic, explore risk factors associated with infection, and investigate the impact of antibody titres on assay sensitivity. Methods: HCWs at Sheffield Teaching Hospitals NHS Foundation Trust were prospective...

Background: We aimed to measure SARS-CoV-2 seroprevalence in a cohort of healthcare workers (HCWs) during the first UK wave of the COVID-19 pandemic, explore risk factors associated with infection, and investigate the impact of antibody titres on assay sensitivity. Methods: HCWs at Sheffield Teaching Hospitals NHS Foundation Trust were prospective...

The strong humoral immune response produced against the SARS-CoV-2 nucleocapsid (N) and spike (S) proteins has underpinned serological testing but the prevalence of antibody responses to other SARS-CoV-2 proteins, which may be of use as further serological markers, is still unclear. Cell-based serological screening platforms can fulfil a crucial ni...

Background: We aimed to measure SARS-CoV-2 seroprevalence in a cohort of healthcare workers (HCWs) during the first UK wave of the COVID-19 pandemic, explore risk factors associated with infection, and investigate the impact of antibody titres on assay sensitivity. Methods: HCWs at Sheffield Teaching Hospitals NHS Foundation Trust were prospectivel...

Background: We aimed to measure SARS-CoV-2 seroprevalence in a cohort of healthcare workers (HCWs) during the first UK wave of the COVID-19 pandemic, explore risk factors associated with infection, and investigate the impact of antibody titres on assay sensitivity. Methods: HCWs at Sheffield Teaching Hospitals NHS Foundation Trust (STH) were prospe...

The Schistosoma mansoni cercarial elastase (SmCE) has previously been shown to be poorly immunogenic in mice. However, a minority of mice were able to produce antibodies against SmCE after multiple immunizations with crude preparations containing the enzyme. These mice were partially protected against challenge infections of S. mansoni . In the pre...

Helicases catalyze the unwinding of double-stranded nucleic acids where structure and phosphate backbone contacts, rather than nucleobase sequence, usually determines substrate specificity. We have expressed and purified a putative helicase encoded by the D10 gene of bacteriophage T5. Here we report that this hitherto uncharacterized protein posses...

Acromegaly is a human disease of growth hormone (GH) excess with considerable morbidity and increased mortality. Somatostatin analogues are first line medical treatment but the disease remains uncontrolled in up to 40% of patients. GH receptor (GHR) antagonist therapy is more effective but requires frequent high-dose injections. We have developed a...

Background: The UK acromegaly register reported that <60% of acromegalics on medical therapy had controlled disease (1). Pegvisomant, a growth hormone antagonist (GHA), controls disease in >95% cases, but is not cost-effective and requires high dose daily injections (2). We have developed a fusion technology for making a cost-effective long-acting...

ECE 2016 Meeting abstract.

Maintenance of genome integrity requires that branched nucleic acid molecules are accurately processed to produce double-helical DNA. Flap endonucleases are essential enzymes that trim such branched molecules generated by Okazaki fragment synthesis during replication. Here, we report crystal structures of bacteriophage T5 flap endonuclease in compl...

Escherichia coli Exonuclease IX (ExoIX), encoded by the xni gene, was the first identified member of a novel subfamily of ubiquitous flap endonucleases (FENs), which possess only one of the two catalytic metal-binding sites characteristic of other FENs. We have solved the first structure of one of these enzymes, that of ExoIX itself, at high resolu...

Background: The development of recombinant biologics has had a major impact on many diseases. However, most biologics are rapidly cleared from the body and therefore require frequent injection regimens. There is therefore a need for technologies that allow the half-lives of these molecules to be extended in a predictable manner. Hypothesis Increasi...

Bacteriophage T5 has a 120 kb double-stranded linear DNA genome encoding most of the genes required for its own replication. This lytic bacteriophage has a burst size of ∼500 new phage particles per infected cell, demonstrating that it is able to turn each infected bacterium into a highly efficient DNA manufacturing machine. To begin to understand...

Introduction The GHLR-Fusions Expression and Purification Analysis of the LR-Fusions LR-Fusions: The Next Generation in Hormone Treatment Conclusion References

We have investigated the interaction between GH (growth hormone) and GHR (GH receptor). We previously demonstrated that a truncated GHR that possesses a transmembrane domain but no cytoplasmic domain blocks receptor signalling. Based on this observation we investigated the impact of tethering the receptor's extracellular domain to the cell surface...

We have investigated the interaction between growth hormone (GH) and its receptor (GHR). We previously demonstrated that a truncated growth hormone receptor that possesses a transmembrane domain but no cytoplasmic domain blocks receptor signaling. Based on this observation we investigated the impact of tethering the receptor's extracellular domain...

Hormone therapies have been used since the early 20th Century and belong to a group of drugs that has recently become known as 'biologics'. Biologics are medicinal products that have been produced by biological processes as opposed to chemically synthesized drugs. The term biologics spans a wide range of products that include therapeutics such as o...

Conference poster

Flap endonucleases (FENs) are divalent metal ion-dependent phosphodiesterases. Metallonucleases are often assigned a “two-metal ion mechanism” where both metals contact the scissile phosphate diester. The spacing of the two metal ions observed in T5FEN structures appears to preclude this mechanism. However, the overall reaction catalyzed by wild ty...

A typical result for the trace of DNA relative to the DNA polymerase. U0 = 16 kBT. (TIF)

Time distributions of the 5′-nuclease domain transiting from the inactive to active modes. (A) d = 2 nm. (B) d = 2.5 nm. (TIF)

Schematic diagram to illustrate the external load Fload acting on the residues (blue dots) of the 5′-nuclease domain. (A) Equilibrium position of the 5′-nuclease domain relative to the polymerase domain. (B) A transient position of the 5′-nuclease domain. (TIF)

Forms of interaction potential of the polymerase domain and the 5′-nuclease domain with the flap DNA substrate, U (x, 0, 0), U (0, y, 0) and U (0, 0, z), with A = 0.5 nm and U0 = kBT. (TIF)

A typical result for the trace of DNA relative to the DNA polymerase. U0 = 16 kBT. (TIF)

Calculated results of the mean transition time Tm versus the spring constant K for different values of d. Dotted line corresponds to K = 8.56 pN/nm. V0 = 18 kBT (TIF)

Temperature dependence of viscosity. (DOC)

Experimentally observed x-ray structure of Taq polymerase based on 1TAQ.pdb. Thumb (light blue), palm (grey), fingers (green), proofreading domain (purple), and 5′ -nuclease domain (dark blue) are shown as a backbone cartoon rendered using Pymol (DeLano Scientific). An oversized grey sphere marks the active site of the 5′-nuclease domain, while red...

A typical result for the trace of DNA relative to the DNA polymerase. U0 = 16 kBT. (TIF)

A typical result for the trace of DNA relative to the DNA polymerase. U0 = 16 kBT. (TIF)

Another form of the interaction potential of the polymerase domain and the 5′-nuclease domain with the flap DNA substrate, with U (x, 0, 0), U (0, y, 0) and U (0, 0, z) being shown in (A), (B) and (C), respectively. U0 = kBT. (TIF)

Calculated results of the mean time Tm for the 5′-nuclease domain to transit from the inactive to active modes as a function of the interaction strength V0 between the 5′-nuclease domain and the flap DNA substrate, with d = 2 nm. (TIF)

Bacteria contain DNA polymerase I (PolI), a single polypeptide chain consisting of ∼930 residues, possessing DNA-dependent DNA polymerase, 3'-5' proofreading and 5'-3' exonuclease (also known as flap endonuclease) activities. PolI is particularly important in the processing of Okazaki fragments generated during lagging strand replication and must u...

A fundamental concern for all new biological therapeutics is the possibility of inducing an immune response. We have recently demonstrated that an LR-fusion (ligand-receptor fusion) of growth hormone generates a potent long-acting agonist; however, the immunogenicity and toxicity of these molecules have not been tested. To address these issues, we...

During replication and repair flap endonucleases (FENs) catalyze endonucleolytic and exonucleolytic (EXO) DNA hydrolyses. Altering the leaving group pK(a), by replacing the departing nucleoside with analogues, had minimal effect on k(cat)/K(M) in a T5FEN-catalyzed EXO reaction, producing a very low Brønsted coefficient, β(lg). Investigation of the...

FENs (flap endonucleases) play essential roles in DNA replication, pivotally in the resolution of Okazaki fragments. In eubacteria, DNA PolI (polymerase I) contains a flap processing domain, the N-terminal 5'-->3' exonuclease. We present evidence of paralogous FEN-encoding genes present in many eubacteria. Two distinct classes of these independent...

Protein nucleases and RNA enzymes depend on divalent metal ions to catalyze the rapid hydrolysis of phosphate diester linkages of nucleic acids during DNA replication, DNA repair, RNA processing, and RNA degradation. These enzymes are widely proposed to catalyze phosphate diester hydrolysis using a "two-metal-ion mechanism." Yet, analyses of flap e...

Early in the Spring of 2000, I attended a series of increasingly animated and exciting meetings with Richard Ross, a clinician with a passion for research specializing in endocrinology, and Pete Artymiuk, an equally enthusiastic structural biologist working in Sheffield University's Krebs Institute.

Osteoprotegerin (OPG) binds the ligand for receptor activator of nuclear factor kappaB (RANKL) to prevent association with its receptor RANK and inhibit osteoclast-mediated bone resorption. OPG has been reported, recently, to inhibit tumor necrosis factor-related apoptosis-induced ligand (TRAIL)-induced tumor cell apoptosis. This raises the possibi...

Osteoprotegerin (OPG) binds the ligand for receptor activator of nuclear factor κB (RANKL) to prevent association with its receptor RANK and inhibit osteoclast-mediated bone resorption. OPG has been reported, recently, to inhibit tumor necrosis factor-related apoptosis-induced ligand (TRAIL)-induced tumor cell apoptosis. This raises the possibility...

Cytokine hormones have a short plasma half-life and require frequent administration. For example, growth hormone replacement involves daily injections. In common with other cytokines, the extracellular domain of the growth hormone receptor circulates as a binding protein, which naturally prolongs the biological half-life of growth hormone. Here we...

Flap endonucleases (FENs) catalyse the exonucleolytic hydrolysis of blunt-ended duplex DNA substrates and the endonucleolytic cleavage of 5'-bifurcated nucleic acids at the junction formed between single and double-stranded DNA. The specificity and catalytic parameters of FENs derived from T5 bacteriophage and Archaeoglobus fulgidus were studied wi...

In the modern biochemistry laboratory, we take for granted the ability to determine the concentration of DNA quickly and accurately. Commercial kits, high-throughput screening and sophisticated single-photon detection systems would be unrecognizable to the pioneers of the early DNA assays, but, even today, their contributions are still being acknow...

The flap endonucleases (FENs) participate in a wide range of processes involving the structure-specific cleavage of branched nucleic acids. They are also able to hydrolyse DNA and RNA substrates from the 5′-end, liberating mono-, di- and polynucleotides terminating with a 5′ phosphate. Exonuclease IX is a paralogue of the small fragment of Escheric...

The flap endonucleases (FENs) participate in a wide range of processes involving the structure-specific cleavage of branched nucleic acids. They are also able to hydrolyse DNA and RNA substrates from the 50-end, liberating mono-, di- and polynucleotides terminating with a 50 phosphate. Exonuclease IX is a paralogue of the small fragment of Escheric...

Respiratory pathogens, such as Neisseria meningitidis, secrete site-specific proteases able to cleave human immunoglobulin A1 (IgA1), the first line of defense at mucosal membranes. Bacterial isolates show wide variability in IgA1 protease activity, and those isolated from patients with clinical infection possess the highest levels of activity. A f...

Running Title: Binding affinity of OPG for RANKL and TRAIL SUMMARY Osteoprotegerin (OPG) binds the ligand for receptor activator of nuclear factor kappa B (RANKL) to prevent association with its receptor RANK and inhibit osteoclast-mediated bone resorption. OPG has been reported, recently, to inhibit TNF-related apoptosis-induced ligand (TRAIL)-ind...

Several features of phage type 5 make this a very interesting and rather unusual lytic bacteriophage. Phage T5 does not encode an RNA polymerase, unlike the T7 phage, nor does it contain unusual DNA modifications such as the 5-hydroxymethyl deoxycytidine present in phage T4. T5 DNA carries some of the strongest known prokaryotic promoters but they...

The coevolution of humans and infectious agents has exerted selective pressure on the immune system to control potentially lethal infections. Correspondingly, pathogens have evolved with various strategies to modulate and circumvent the host's innate and adaptive immune response. Schistosoma species are helminth parasites with genes that have been...

The coevolution of humans and infectious agents has exerted selective pressure on the immune system to control potentially lethal infections. Correspondingly, pathogens have evolved with various strategies to modulate and circumvent the host's innate and adaptive immune response. Schistosoma species are helminth parasites with genes that have been...

The birth of bacterial genomics since the mid-1990s brought withit several conceptual modifications and wholly new controversies. Working beyond the scope of the neo-Darwinian evolutionary synthesis, a group of leading microbial evolutionists addresses the following and related issues, often with markedly varied viewpoints: · Did the eukaryotic nuc...

IgA1 (immunoglobulin A1) antibodies are the first line of defence against microbial pathogens such as Neisseria meningitidis and Haemophilus influenzae. However, these bacteria secrete a site-specific protease that is capable of cleaving human IgA1 and interacting with other host components. The IgA proteases are released by the type V secretion pa...

Infectious pathogens can selectively stimulate activation or suppression of T cells to facilitate their survival within humans. In this study we demonstrate that the trematode parasite Schistosoma mansoni has evolved with two distinct mechanisms to suppress T cell activation. During the initial 4- to 12-wk acute stages of a worm infection both CD4(...

Flap endonucleases (FENs) have essential roles in DNA processing. They catalyze exonucleolytic and structure-specific endonucleolytic DNA cleavage reactions. Divalent metal ions are essential cofactors in both reactions. The crystal structure of FEN shows that the protein has two conserved metal-binding sites. Mutations in site I caused complete lo...

On the basis of structural work, metal ions are proposed to play a catalytic role in reactions mediated by many phosphoryl transfer enzymes. To gain dynamic support for such mechanisms, the role of metal ion cofactors in phosphate diester hydrolysis catalysed by a flap endonuclease has been studied. The pH maximal rate profiles were measured in the...

The flap endonucleases, or 5' nucleases, are involved in DNA replication and repair. They possess both 5'-3' exonucleolytic activity and the ability to cleave bifurcated, or branched DNA, in an endonucleolytic, structure-specific manner. These enzymes share a great degree of structural and sequence similarity. Conserved acidic amino acids, whose pr...

Previous structural studies on native T5 5' nuclease, a member of the flap endonuclease family of structure-specific nucleases, demonstrated that this enzyme possesses an unusual helical arch mounted on the enzyme's active site. Based on this structure, the protein's surface charge distribution, and biochemical analyses, a model of DNA binding was...

Nontypeable Haemophilus influenzae strains form part of the normal flora of the human upper respiratory tract but are also implicated in a wide range of diseases. Infections caused by nontypeable H influenzae are major health and socioeconomic burdens. No single bacterial trait has been associated with disease as opposed to colonization. To compare...

The Cambridge Healthtech Institute's second international conference entitled "Recombinant Therapeutics: Antibodies and Biomolecules" was held in Baltimore, MD, USA, 30 May-1 June 2001.

Mounted on the Internet with the permission of The Biochemical Society (2001). A new method for continuous biopanning has been developed. We have combined the power of affinity chromatography with the fecundity of bacteria in a unique process that mimics clonal selection. Mixed populations of bacteria were applied to a fermenter containing the immo...

T5 5'-3' exonuclease is a member of a homologous group of 5' nucleases which require divalent metal co-factors. Structural and biochemical studies suggest that single-stranded DNA substrates thread through a helical arch or hole in the protein, thus bringing the phosphodiester backbone into close proximity with the active site metal co-factors. In...

The Biochemical Society colloquium on New Frontiers in DNA Repair was held on 19–20 December 2000 in Sussex, UK.

T5 5'-3'-exonuclease is a member of a family of homologous 5'-nucleases essential for DNA replication and repair. We have measured the variation of the steady state parameters of the enzyme with pH. The log of the association constant of the enzyme and substrate is pH-independent between pH 5 and 7, but at higher pH, it decreases (gradient -0.91 +/...

T5 5′-3′-exonuclease is a member of a family of homologous 5′-nucleases essential for DNA replication and repair. We have measured the variation of the steady state parameters of the enzyme with pH. The log of the association constant of the enzyme and substrate is pH-independent between pH 5 and 7, but at higher pH, it decreases (gradient −0.91 ±...

Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae possess the ability to cleave human IgA1 antibodies, and all successfully colonize and occasionally invade the human upper respiratory tract. N. meningitidis invades the bloodstream after a period of nasopharyngeal colonization. We directly compared levels of IgA1 protease...

Bacteriophage T5 5′→3′ exonuclease is a member of a family of sequence related 5′-nucleases which play an essential role in DNA replication. The 5′-nucleases have both exonucleolytic and structure-specific endonucleolytic DNA cleavage activity and are conserved in organisms as diverse as bacteriophage and mammals. Here, we report the development of...

Efficient cellular DNA replication requires the activity of a 5'-3' exonuclease. These enzymes are able to hydrolyze DNA.DNA and RNA.DNA substrates exonucleolytically, and they are structure-specific endonucleases. The 5'-3' exonucleases are conserved in organisms as diverse as bacteriophage and mammals. Crystal structures of three representative e...

Many processes, such as DNA replication, recombination and repair, produce branched DNA structures. These bifurcated molecules are trimmed by a group of homologous 5'-3' exonucleases (also known as 5' nucleases) in structure-specific cleavage reactions. X-ray crystallographical and biochemical studies suggest that ssDNA substrates become threaded t...

The crystal structure of an archaeal 5' flap endonuclease suggests a common mode of action in this divergent group of essential replicative enzymes.

The three dimensional crystal structure of T5 5′-3′ exonuclease was compared with that of two other members of the 5′-3′ exonuclease family: T4 ribonuclease H and the N-terminal domain of Thermus aquaticus DNA polymerase I. Though these structures were largely similar, some regions of these enzymes show evidence of significant molecular flexibility...

Phage T5 exonuclease is a 5′→3′ exodeoxyribonuclease that also exhibits endonucleolytic activity on flap structures (branched duplex DNA containing a free single-stranded 5′-end). Oligonucleotides were used to construct duplexes with either blunt ends, 5′-over-hangs, 3′-overhangs, a flap or a forked end (pseudo-Y). The binding of T5 exonuclease to...