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8 results on '"Samar Hodeib"'

1. Magnetic Tweezers-Based Single-Molecule Assays to Study Interaction of E. coli SSB with DNA and RecQ Helicase

Author

Debjani, Bagchi, Weiting, Zhang, Samar, Hodeib, Bertrand, Ducos, Vincent, Croquette, and Maria, Manosas

Subjects
DNA, Bacterial, DNA-Binding Proteins, Time Factors, RecQ Helicases, Escherichia coli Proteins, Magnetic Phenomena, Escherichia coli, Single Molecule Imaging, Protein Binding
Abstract

The ability of magnetic tweezers to apply forces and measure molecular displacements has resulted in its extensive use to study the activity of enzymes involved in various aspects of nucleic acid metabolism. These studies have led to the discovery of key aspects of protein-protein and protein-nucleic acid interaction, uncovering dynamic heterogeneities that are lost to ensemble averaging in bulk experiments. The versatility of magnetic tweezers lies in the possibility and ease of tracking multiple parallel single-molecule events to yield statistically relevant single-molecule data. Moreover, they allow tracking both fast millisecond dynamics and slow processes (spanning several hours). In this chapter, we present the protocols used to study the interaction between E. coli SSB, single-stranded DNA (ssDNA), and E. coli RecQ helicase using magnetic tweezers. In particular, we propose constant force and force modulation assays to investigate SSB binding to DNA, as well as to characterize various facets of RecQ helicase activity stimulation by SSB.

Published
2021

2. Folding and Persistence Time of Intramolecular G-Quadruplexes Transiently Embedded in a DNA duplex

Author

Patrizia Alberti, Jimmy Ouellet, Jean-François Allemand, Alexandra Joubert, Phong Lan Thao Tran, Jean-Baptiste Boulé, Martin Rieu, Anthony Bugaut, Vincent Croquette, Samar Hodeib, Structure et Instabilité des Génomes (STRING), Muséum national d'Histoire naturelle (MNHN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie de l'ENS Paris (IBENS), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), ABCD : Biophysique des Biomolécules, Laboratoire de physique de l'ENS - ENS Paris (LPENS), Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)-Sorbonne Université (SU)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)-Sorbonne Université (SU)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), Institut de biologie de l'ENS Paris (UMR 8197/1024) (IBENS), Département de Biologie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de physique de l'ENS - ENS Paris (LPENS (UMR_8023)), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)-École normale supérieure - Paris (ENS Paris), and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)

Subjects
0303 health sciences, Chemistry, [SDV]Life Sciences [q-bio], DNA replication, 010402 general chemistry, G-quadruplex, Origin of replication, 01 natural sciences, 0104 chemical sciences, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Folding (chemistry), 03 medical and health sciences, chemistry.chemical_compound, Transcription (biology), Complementary DNA, Biophysics, A-DNA, DNA, 030304 developmental biology
Abstract

G-quadruplex (G4) DNA structures have emerged as important regulatory elements during DNA replication, transcription or repair. While manyin-vitrostudies have focused on the kinetics of G4 formation within DNA single-strands, G4 are foundin-vivoin double-stranded DNA regions, where their formation is challenged by pairing between the two complementary strands. Since the energy of hybridization of Watson-Crick structures dominates the energy of G4 folding, this competition should play a critical role on the persistence of G4in vivo. To address this issue, we designed a single molecule assay allowing measuring G4 folding and persistence while the structure is periodically challenged by the complementary strand. We quantified both the folding rate and the persistence time of biologically relevant G4 structures and showed that the dynamics of G4 formation depends strongly on the genomic location. G4 are found much more stable in promoter regions and replication origins than in telomeric regions. In addition, we characterized how G4 dynamics was affected by G4 ligands and showed that both folding rate and persistence increased. Our assay opens new perspectives for the measurement of G4 dynamics, which is critical to understand their role in genetic regulation.

Published
2021
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4. Magnetic Tweezers-Based Single-Molecule Assays to Study Interaction of E. coli SSB with DNA and RecQ Helicase

Author

Vincent Croquette, Debjani Bagchi, Bertrand Ducos, Maria Manosas, Samar Hodeib, and Weiting Zhang

Subjects
0303 health sciences, Magnetic tweezers, RecQ helicase, Ensemble averaging, Nucleic acid metabolism, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, chemistry, Biophysics, Molecule, Constant force, 030217 neurology & neurosurgery, DNA, 030304 developmental biology
Abstract

The ability of magnetic tweezers to apply forces and measure molecular displacements has resulted in its extensive use to study the activity of enzymes involved in various aspects of nucleic acid metabolism. These studies have led to the discovery of key aspects of protein-protein and protein-nucleic acid interaction, uncovering dynamic heterogeneities that are lost to ensemble averaging in bulk experiments. The versatility of magnetic tweezers lies in the possibility and ease of tracking multiple parallel single-molecule events to yield statistically relevant single-molecule data. Moreover, they allow tracking both fast millisecond dynamics and slow processes (spanning several hours). In this chapter, we present the protocols used to study the interaction between E. coli SSB, single-stranded DNA (ssDNA), and E. coli RecQ helicase using magnetic tweezers. In particular, we propose constant force and force modulation assays to investigate SSB binding to DNA, as well as to characterize various facets of RecQ helicase activity stimulation by SSB.

Published
2021
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5. Single molecule kinetics uncover roles for E. coli RecQ DNA helicase domains and interaction with SSB

Author

James L. Keck, Vincent Croquette, Debjani Bagchi, Bertrand Ducos, Weiting Zhang, Kelly A. Manthei, Maria Manosas, Samar Hodeib, Universitat de Barcelona (UB), Centro Investigacion Biomedica en Red Bioingenieria, Biomateriales y Nanomedicina - CIBER-BBN (SPAIN), Institut de biologie de l'ENS Paris (IBENS), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Physique Statistique de l'ENS (LPS), Fédération de recherche du Département de physique de l'Ecole Normale Supérieure - ENS Paris (FRDPENS), Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Diderot - Paris 7 (UPD7)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Sorbonne Université - Faculté de Médecine (SU FM), Sorbonne Université (SU), Université Paris Diderot - Paris 7 (UPD7), Université Paris Diderot - Paris 7 (UPD7)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Fédération de recherche du Département de physique de l'Ecole Normale Supérieure - ENS Paris (FRDPENS), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), Institut de biologie de l'ENS Paris (UMR 8197/1024) (IBENS), Département de Biologie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)

Subjects
DNA, Bacterial, Models, Molecular, 0301 basic medicine, congenital, hereditary, and neonatal diseases and abnormalities, Optical Tweezers, Protein Conformation, Inverted Repeat Sequences, [SDV]Life Sciences [q-bio], Protein domain, Biology, medicine.disease_cause, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Protein structure, Protein Domains, Escherichia coli, Genetics, medicine, RNase D, [SDV.GEN]Life Sciences [q-bio]/Genetics, RecQ Helicases, Escherichia coli Proteins, nutritional and metabolic diseases, Helicase, Processivity, Single Molecule Imaging, Cell biology, DNA-Binding Proteins, Kinetics, enzymes and coenzymes (carbohydrates), 030104 developmental biology, chemistry, biology.protein, Gene Deletion, 030217 neurology & neurosurgery, DNA
Abstract

International audience; Most RecQ DNA helicases share a conserved domain arrangement that mediates their activities in genomic stability. This arrangement comprises a helicase motor domain, a RecQ C-terminal (RecQ-C) region including a winged-helix (WH) domain, and a 'Helicase and RNase D C-terminal' (HRDC) domain. Single-molecule real-time translocation and DNA unwinding by full-length Escherichia coli RecQ and variants lacking either the HRDC or both the WH and HRDC domains was analyzed. RecQ operated under two interconvertible kinetic modes, 'slow' and 'normal', as it unwound duplex DNA and translocated on single-stranded (ss) DNA. Consistent with a crystal structure of bacterial RecQ bound to ssDNA by base stacking, abasic sites blocked RecQ unwinding. Removal of the HRDC domain eliminates the slow mode while preserving the normal mode of activity. Unexpectedly, a RecQ variant lacking both the WH and HRDC domains retains weak helicase activity. The inclusion of E. coli ssDNA-binding protein (SSB) induces a third 'fast' unwinding mode four times faster than the normal RecQ mode and enhances the overall helicase activity (affinity, rate, and processivity). SSB stimulation was, furthermore, observed in the RecQ deletion variants, including the variant missing the WH domain. Our results support a model in which RecQ and SSB have multiple interacting modes.

Published
2018
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6. A mechanistic study of helicases with magnetic traps

Author

Samar Hodeib, Joanne Kanaan, Jean-François Allemand, Vincent Croquette, Debjani Bagchi, Saurabh Raj, Bertrand Ducos, David Bensimon, Francesca Fiorini, Hervé Le Hir, Maria Manosas, and Weiting Zhang

Subjects
0301 basic medicine, Genetics, DNA clamp, 030102 biochemistry & molecular biology, biology, DNA replication, Helicase, Eukaryotic DNA replication, Biochemistry, RNA Helicase A, Cell biology, 03 medical and health sciences, chemistry.chemical_compound, 030104 developmental biology, chemistry, biology.protein, Primase, Molecular Biology, Polymerase, DNA
Abstract

Helicases are a broad family of enzymes that separate nucleic acid double strand structures (DNA/DNA, DNA/RNA or RNA/RNA) and thus are essential to DNA replication and the maintenance of nucleic acid integrity. We review the picture that has emerged from single molecule studies of the mechanisms of DNA and RNA helicases and their interactions with other proteins. Many features have been uncovered by these studies that were obscured by bulk studies, such as DNA strands switching, mechanical (rather than biochemical) coupling between helicases and polymerases, helicase-induced re-hybridization and stalled fork rescue. This article is protected by copyright. All rights reserved.

Published
2017
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7. Single molecule studies of helicases with magnetic tweezers

Author

Samar Hodeib, Jean-François Allemand, Vincent Croquette, Debjani Bagchi, Weiting Zhang, Maria Manosas, Saurabh Raj, David Bensimon, and Bertrand Ducos

Subjects
DNA Replication, 0301 basic medicine, Magnetic tweezers, Optical Tweezers, High resolution, Nanotechnology, Biology, General Biochemistry, Genetics and Molecular Biology, Magnetics, 03 medical and health sciences, chemistry.chemical_compound, Molecule, Molecular Biology, chemistry.chemical_classification, DNA, Cruciform, DNA Helicases, DNA replication, RNA, Helicase, DNA, Single Molecule Imaging, 030104 developmental biology, Enzyme, chemistry, biology.protein, Biophysics
Abstract

Helicases are a broad family of enzymes that perform crucial functions in DNA replication and in the maintenance of DNA and RNA integrity. A detailed mechanical study of helicases on DNA and RNA is possible using single molecule manipulation methods. Among those, magnetic tweezers (or traps) present a convenient, moderate throughput assay (tens of enzymes can be monitored simultaneously) that allow for high resolution (single base-pair) studies of these enzymes in various conditions and on various substrates (double and single stranded DNA and RNA). Here we discuss various implementation of the basic assay relevant for these studies.

Published
2016
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