ORCID Profile
0000-0002-4533-3816
Current Organisation
University of York
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Publisher: Cold Spring Harbor Laboratory
Date: 10-05-2022
DOI: 10.1101/2022.05.10.491410
Abstract: Many essential cellular processes rely on substrate rotation or translocation by a multi-subunit, ring-type NTPase. A large number of double-stranded DNA viruses, including tailed bacteriophages and herpes viruses, use a homomeric ring ATPase to processively translocate viral genomic DNA into procapsids during assembly. Our current understanding of viral DNA packaging comes from three archetypal bacteriophage systems: cos, pac and phi29. Detailed mechanistic understanding exists for pac and phi29, but not for cos . Here we reconstituted in vitro a cos packaging system based on bacteriophage HK97 and provided a detailed biochemical and structural description. We used a photobleaching-based, single-molecule assay to determine the stoichiometry of the DNA-translocating ATPase large terminase. Crystal structures of the large terminase and DNA-recruiting small terminase, a first for both this phage and a cos system, reveal unexpected mechanistic similarities between cos and pac systems. At the same time, mutational and biochemical analyses indicate a new regulatory mechanism for ATPase multimerization and coordination in the HK97 system. This work therefore establishes a framework for studying the evolutionary relationships between ATP-dependent DNA translocation machineries in double-stranded DNA viruses.
Publisher: Cold Spring Harbor Laboratory
Date: 24-02-2023
DOI: 10.1101/2023.02.24.529869
Abstract: Double-stranded DNA viruses utilise machinery, made of terminase proteins, to package viral DNA into the capsid. For cos bacteriophage, a defined signal, recognised by small terminase, flanks each genome unit. Here we present the first structural data for a cos virus DNA packaging motor, assembled from the bacteriophage HK97 terminase proteins, procapsids encompassing the portal protein, and DNA containing a cos site. The cryo-EM structure is consistent with the packaging termination state adopted after DNA cleavage, with DNA density within the large terminase assembly ending abruptly at the portal protein entrance. Retention of the large terminase complex after cleavage of the short DNA substrate suggests that motor dissociation from the capsid requires headful pressure, in common with pac viruses. Interestingly, the clip domain of the 12-subunit portal protein does not adhere to C 12 symmetry, indicating asymmetry induced by binding of the large terminase/DNA. The motor assembly is also highly asymmetric, showing a ring of 5 large terminase monomers, tilted against the portal. Variable degrees of extension between N- and C-terminal domains of in idual subunits suggest a mechanism of DNA translocation driven by inter-domain contraction and relaxation.
Publisher: International Union of Crystallography (IUCr)
Date: 22-02-2012
Publisher: International Union of Crystallography (IUCr)
Date: 30-06-2015
DOI: 10.1107/S1399004715009220
Abstract: The reduction of uridine to dihydrouridine at specific positions in tRNA is catalysed by dihydrouridine synthase (Dus) enzymes. Increased expression of human dihydrouridine synthase 2 (hDus2) has been linked to pulmonary carcinogenesis, while its knockdown decreased cancer cell line viability, suggesting that it may serve as a valuable target for therapeutic intervention. Here, the X-ray crystal structure of a construct of hDus2 encompassing the catalytic and tRNA-recognition domains (residues 1–340) determined at 1.9 Å resolution is presented. It is shown that the structure can be determined automatically by phenix.mr_rosetta starting from a bacterial Dus enzyme with only 18% sequence identity and a significantly ergent structure. The overall fold of the human Dus2 is similar to that of bacterial enzymes, but has a larger recognition domain and a unique three-stranded antiparallel β-sheet insertion into the catalytic domain that packs next to the recognition domain, contributing to domain–domain interactions. The structure may inform the development of novel therapeutic approaches in the fight against lung cancer.
Publisher: Cold Spring Harbor Laboratory
Date: 24-02-2023
DOI: 10.1101/2023.02.24.528798
Abstract: Visible surface films, termed slicks, can extensively cover the sea surface, particularly in coastal regions. The sea-surface microlayer (SML), the upper 1-mm at the air-water interface in slicks (slick SML) harbors a distinctive bacterial community, but little is known about SML viruses. Using flow cytometry, metagenomics, and cultivation, we investigated viruses and the bacterial community from a brackish slick SML in comparison to non-slick SML as well as the seawater below (SSW). We conducted size-fractionated filtration of all s les to distinguish viral attachment to hosts and particles. The slick SML contained higher abundances of virus-like particles, prokaryotic cells, and dissolved organic carbon compared to non-slick SML and SSW. The community of 428 viral operational taxonomic units (vOTUs), 426 predicted as lytic, distinctly differed across all size fractions in the slick SML compared to non-slick SML and SSW. The distinctness was underlined by specific metabolic profiles of bacterial metagenome assembled genomes and isolates, which revealed prevalence of motility genes and ersity of CAZymes in the slick SML. Despite overall lower ersity, several vOTUs were enriched in slick SML over slick SSW. Nine vOTUs were only found in slick SML and six of them were targeted by slick SML-specific CRISPR spacers likely originating from Gammaproteobacteria. Moreover, isolation of three previously unknown lytic phages for Alishewanella sp. and Pseudoalteromonas tunicata , representing abundant and actively replicating slick SML bacteria, suggests that viral activity in slicks can contribute to biogeochemical cycling in coastal ecosystems.
Publisher: Proceedings of the National Academy of Sciences
Date: 22-04-2015
Abstract: RNA-binding proteins use erse mechanisms for generating specificity toward distinct RNA molecules. Different subfamilies of bacterial dihydrouridine synthases (Dus) modify specific uridines in tRNA, but the mechanism for selection of the target nucleotide is unknown. We solved crystal structures of the U16-specific Dus from Escherichia coli complexed with two different tRNAs. These structures reveal that the tRNA is bound in a completely different orientation from that observed in a U20-specific enzyme. The major reorientation of the substrate tRNA, driven by unique amino acid “binding signatures” and plasticity in the position of the C-terminal recognition domain, appears to be an evolutionary innovation to the known strategies that define specificity of enzymes toward tRNA.
Publisher: Oxford University Press (OUP)
Date: 09-06-2023
DOI: 10.1093/NAR/GKAD480
Abstract: Double-stranded DNA viruses utilise machinery, made of terminase proteins, to package viral DNA into the capsid. For cos bacteriophage, a defined signal, recognised by small terminase, flanks each genome unit. Here we present the first structural data for a cos virus DNA packaging motor, assembled from the bacteriophage HK97 terminase proteins, procapsids encompassing the portal protein, and DNA containing a cos site. The cryo-EM structure is consistent with the packaging termination state adopted after DNA cleavage, with DNA density within the large terminase assembly ending abruptly at the portal protein entrance. Retention of the large terminase complex after cleavage of the short DNA substrate suggests that motor dissociation from the capsid requires headful pressure, in common with pac viruses. Interestingly, the clip domain of the 12-subunit portal protein does not adhere to C12 symmetry, indicating asymmetry induced by binding of the large terminase/DNA. The motor assembly is also highly asymmetric, showing a ring of 5 large terminase monomers, tilted against the portal. Variable degrees of extension between N- and C-terminal domains of in idual subunits suggest a mechanism of DNA translocation driven by inter-domain contraction and relaxation.
Publisher: Oxford University Press (OUP)
Date: 03-08-2015
DOI: 10.1093/NAR/GKV778
Publisher: Oxford University Press (OUP)
Date: 04-2024
DOI: 10.1093/NAR/GKAC647
Abstract: Many essential cellular processes rely on substrate rotation or translocation by a multi-subunit, ring-type NTPase. A large number of double-stranded DNA viruses, including tailed bacteriophages and herpes viruses, use a homomeric ring ATPase to processively translocate viral genomic DNA into procapsids during assembly. Our current understanding of viral DNA packaging comes from three archetypal bacteriophage systems: cos, pac and phi29. Detailed mechanistic understanding exists for pac and phi29, but not for cos. Here, we reconstituted in vitro a cos packaging system based on bacteriophage HK97 and provided a detailed biochemical and structural description. We used a photobleaching-based, single-molecule assay to determine the stoichiometry of the DNA-translocating ATPase large terminase. Crystal structures of the large terminase and DNA-recruiting small terminase, a first for a biochemically defined cos system, reveal mechanistic similarities between cos and pac systems. At the same time, mutational and biochemical analyses indicate a new regulatory mechanism for ATPase multimerization and coordination in the HK97 system. This work therefore establishes a framework for studying the evolutionary relationships between ATP-dependent DNA translocation machineries in double-stranded DNA viruses.
Publisher: Oxford University Press (OUP)
Date: 15-12-2015
DOI: 10.1093/NAR/GKV1467
Publisher: Oxford University Press (OUP)
Date: 08-11-2011
DOI: 10.1093/NAR/GKR906
Publisher: International Union of Crystallography (IUCr)
Date: 15-05-2013
Location: United Kingdom of Great Britain and Northern Ireland
Location: Russian Federation
Location: Russian Federation
Location: No location found
Location: No location found
Start Date: 2017
End Date: 2022
Funder: Wellcome Trust
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