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13 results on '"Jameson, Katie H."'

3. Structural and biophysical investigations of two negative regulators of DNA replication initiation in Bacillus subtilis, YabA and SirA

Author

Jameson, Katie H. and Wilkinson, A. J.

Subjects
572.8
Abstract

DNA replication initiation is strictly controlled to maintain chromosome copy number. Over- or under-replication of an organism's genome is detrimental to survival, thus initiation events are tightly regulated to ensure only one round of DNA replication occurs per cell cycle. In prokaryotes, DNA replication commences when a protein initiator, DnaA, forms a helical filament at the origin of replication, oriC, inducing localised DNA unwinding and the recruitment of the replication machinery. The work described here sought to elucidate how DNA replication initiation is regulated in the Gram-positive model organism Bacillus subtilis. In particular, this work aimed to offer insight into the mechanisms of two negative regulators of DNA replication initiation, SirA and YabA; proteins that play significant roles in regulating replication initiation in sporulating and vegetatively growing cells, respectively. Both SirA and YabA interact directly with the initiator DnaA, and YabA has additionally been shown to interact with the DNA polymerase β-clamp, DnaN (an essential component of the replication machinery). Detailed here are structural and biophysical studies of SirA and YabA. This includes the X-ray crystal structure of SirA bound to the N-terminal domain of DnaA in an inhibitory complex, and characterisation of the SirA-DnaADI interface using an in vitro assay. When coupled with in vivo localisation studies carried out by our collaborators, the work on SirA suggests a mechanism for its inhibition of DNA replication. Also detailed are biophysical studies used to characterise the architecture of YabA, guided by the use of in silico models, and the X-ray crystal structure of YabA's N-terminal domain. These results are discussed alongside structural and mutational studies of YabA carried out by our collaborators; collectively the results delineate the full-length structure of YabA and offer insight into its interactions with DnaA and DnaN.

Published
2015

7. Control of Initiation of DNA Replication in Bacillus subtilis and Escherichia coli

Author

Jameson, Katie H. and Wilkinson, Anthony J.

Subjects
oriC, lcsh:Genetics, DnaA, sporulation, lcsh:QH426-470, initiation of DNA replication, regulation of DNA replication, Review, Bacillus subtilis
Abstract

Initiation of DNA Replication is tightly regulated in all cells since imbalances in chromosomal copy number are deleterious and often lethal. In bacteria such as Bacillus subtilis and Escherichia coli, at the point of cytokinesis, there must be two complete copies of the chromosome to partition into the daughter cells following division at mid-cell during vegetative growth. Under conditions of rapid growth, when the time taken to replicate the chromosome exceeds the doubling time of the cells, there will be multiple initiations per cell cycle and daughter cells will inherit chromosomes that are already undergoing replication. In contrast, cells entering the sporulation pathway in B. subtilis can do so only during a short interval in the cell cycle when there are two, and only two, chromosomes per cell, one destined for the spore and one for the mother cell. Here, we briefly describe the overall process of DNA replication in bacteria before reviewing initiation of DNA replication in detail. The review covers DnaA-directed assembly of the replisome at oriC and the multitude of mechanisms of regulation of initiation, with a focus on the similarities and differences between E. coli and B. subtilis.

Published
2017

8. Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners

Author

Felicori, Liza, primary, Jameson, Katie H., additional, Roblin, Pierre, additional, Fogg, Mark J., additional, Garcia-Garcia, Transito, additional, Ventroux, Magali, additional, Cherrier, Mickaël V., additional, Bazin, Alexandre, additional, Noirot, Philippe, additional, Wilkinson, Anthony J., additional, Molina, Franck, additional, Terradot, Laurent, additional, and Noirot-Gros, Marie-Françoise, additional

Published
2015
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11. Structure and interactions of the B acillus subtilis sporulation inhibitor of DNA replication, SirA, with domain I of DnaA.

Author

Jameson, Katie H., Rostami, Nadia, Fogg, Mark J., Turkenburg, Johan P., Grahl, Anne, Murray, Heath, and Wilkinson, Anthony J.

Subjects
*BACILLUS subtilis, *DNA copy number variations, *DNA replication, *BACTERIAL sporulation, *CELL division
Abstract

Chromosome copy number in cells is controlled so that the frequency of initiation of DNA replication matches that of cell division. In bacteria, this is achieved through regulation of the interaction between the initiator protein DnaA and specific DNA elements arrayed at the origin of replication. DnaA assembles at the origin and promotes DNA unwinding and the assembly of a replication initiation complex. SirA is a DnaA-interacting protein that inhibits initiation of replication in diploid B acillus subtilis cells committed to the developmental pathway leading to formation of a dormant spore. Here we present the crystal structure of SirA in complex with the N-terminal domain of DnaA revealing a heterodimeric complex. The interacting surfaces of both proteins are α-helical with predominantly apolar side-chains packing in a hydrophobic interface. Site-directed mutagenesis experiments confirm the importance of this interface for the interaction of the two proteins in vitro and in vivo. Localization of GFP- SirA indicates that the protein accumulates at the replisome in sporulating cells, likely through a direct interaction with DnaA. The SirA interacting surface of DnaA corresponds closely to the HobA-interacting surface of DnaA from H elicobacter pylori even though HobA is an activator of DnaA and SirA is an inhibitor. [ABSTRACT FROM AUTHOR]

Published
2014
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12. Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners

Author

Liza Felicori, Magali Ventroux, Transito Garcia-Garcia, Laurent Terradot, Franck Molina, Anthony J. Wilkinson, Mark J. Fogg, Philippe Noirot, Katie H. Jameson, Alexandre Bazin, Mickaël V. Cherrier, Marie Francoise Noirot-Gros, Pierre Roblin, Sysdiag CNRS Bio-Rad UMR 3145, Cap Delta/Parc Euromédecine, Centre National de la Recherche Scientifique (CNRS), Département Caractérisation et Elaboration des Produits Issus de l'Agriculture (CEPIA), Institut National de la Recherche Agronomique (INRA), York Structural Biology Laboratory, Department of Chemistry, University of York [York, UK], MICrobiologie de l'ALImentation au Service de la Santé (MICALIS), Institut National de la Recherche Agronomique (INRA)-AgroParisTech, Institut de biologie structurale (IBS - UMR 5075), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Informatique, de Modélisation et d'Optimisation des Systèmes (LIMOS), Ecole Nationale Supérieure des Mines de St Etienne-Université Clermont Auvergne (UCA)-Centre National de la Recherche Scientifique (CNRS), Sysdiag-Modélisation et Ingénierie des Systèmes Complexes Biologiques pour le Diagnostic (SysDiag ), BIO-RAD-Centre National de la Recherche Scientifique (CNRS), Institut de biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Ecole Nationale Supérieure des Mines de St Etienne-Université Clermont Auvergne [2017-2020] (UCA [2017-2020])-Centre National de la Recherche Scientifique (CNRS), European integrated project, BaSysBio, Biotechnology and Biological Sciences Research Council, UK, EU-Marie Curie Project AMBER FP7-People [317338], CIBLE program from the region Rhones-alpes, MICALIS INRA, Felicori, Liza, Jameson, Katie H., Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), and Microbiologie moléculaire et biochimie structurale / Molecular Microbiology and Structural Biochemistry (MMSB)

Subjects
Models, Molecular, 0301 basic medicine, Protein Conformation, Amino Acid Motifs, Intracellular Space, dnaN, MESH: DNA Replication, Plasma protein binding, MESH: Amino Acid Sequence, MESH: Zinc, MESH: Amino Acid Motifs, Protein structure, MESH: Structure-Activity Relationship, MESH: Protein Conformation, Structural Biology, Protein Interaction Mapping, MESH: Bacterial Proteins, Genetics, DNA clamp, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Protein Multimerization, DNA-Binding Proteins, Protein Transport, Zinc, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], MESH: Intracellular Space, MESH: Models, Molecular, Bacillus subtilis, Protein Binding, DNA Replication, MESH: Protein Transport, MESH: Mutation, Molecular Sequence Data, MESH: Sequence Alignment, Biology, Structure-Activity Relationship, 03 medical and health sciences, Bacterial Proteins, Position-Specific Scoring Matrices, MESH: Protein Binding, Protein Interaction Domains and Motifs, Amino Acid Sequence, Homology modeling, MESH: Protein Interaction Domains and Motifs, Binding Sites, MESH: Molecular Sequence Data, MESH: Protein Interaction Mapping, DNA replication, MESH: Position-Specific Scoring Matrices, MESH: Bacillus subtilis, MESH: Multiprotein Complexes, DnaA, 030104 developmental biology, MESH: Binding Sites, Replication Initiation, Multiprotein Complexes, Mutation, Biophysics, Protein Multimerization, Sequence Alignment, MESH: DNA-Binding Proteins
Abstract

International audience; YabA negatively regulates initiation of DNA replication in low-GC Gram-positive bacteria. The protein exerts its control through interactions with the initiator protein DnaA and the sliding clamp DnaN. Here, we combined X-ray crystallography, X-ray scattering (SAXS), modeling and biophysical approaches, with in vivo experimental data to gain insight into YabA function. The crystal structure of the N-terminal domain (NTD) of YabA solved at 2.7 Å resolution reveals an extended α-helix that contributes to an intermolecular four-helix bundle. Homology modeling and biochemical analysis indicates that the C-terminal domain (CTD) of YabA is a small Zn-binding domain. Multi-angle light scattering and SAXS demonstrate that YabA is a tetramer in which the CTDs are independent and connected to the N-terminal four-helix bundle via flexible linkers. While YabA can simultaneously interact with both DnaA and DnaN, we found that an isolated CTD can bind to either DnaA or DnaN, individually. Site-directed mutagenesis and yeast-two hybrid assays identified DnaA and DnaN binding sites on the YabA CTD that partially overlap and point to a mutually exclusive mode of interaction. Our study defines YabA as a novel structural hub and explains how the protein tetramer uses independent CTDs to bind multiple partners to orchestrate replication initiation in the bacterial cell.

Published
2016
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13. In vitro Di-ubiquitin Formation Assay and E3 Cooperation Assay.

Author

Burge RJ, Jameson KH, Wilkinson AJ, and Mottram JC

Abstract

Ubiquitination is a post-translational modification conserved across eukaryotic species. It contributes to a variety of regulatory pathways, including proteasomal degradation, DNA repair, and cellular differentiation. The ubiquitination of substrate proteins typically requires three ubiquitination enzymes: a ubiquitin-activating E1, a ubiquitin-conjugating E2, and an E3 ubiquitin ligase. Cooperation between E2s and E3s is required for substrate ubiquitination, but some ubiquitin-conjugating E2s are also able to catalyze by themselves the formation of free di-ubiquitin, independently or in cooperation with a ubiquitin E2 variant. Here, we describe a method for assessing (i) di-ubiquitin formation by an E1 together with an E2 and an E2 variant, and (ii) the cooperation of an E3 with an E1 and E2 (with or without the E2 variant). Reaction products are assessed using western blotting with one of two antibodies: the first detects all ubiquitin conjugates, while the second specifically recognizes K63-linked ubiquitin. This allows unambiguous identification of ubiquitinated species and assessment of whether K63 linkages are present. We have developed these methods for studying ubiquitination proteins of Leishmania mexicana , specifically the activities of the E2, UBC2, and the ubiquitin E2 variant UEV1, but we anticipate the assays to be applicable to other ubiquitination systems with UBC2/UEV1 orthologues., Competing Interests: Competing interests The authors declare no competing interests, (Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC.)

Published
2022
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