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A Molecular Toolbox to Engineer Site-Specific DNA Replication Perturbation

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Genome Instability

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1672))

Abstract

Site-specific arrest of DNA replication is a useful tool for analyzing cellular responses to DNA replication perturbation. The E. coli Tus-Ter replication barrier can be reconstituted in eukaryotic cells as a system to engineer an unscheduled collision between a replication fork and an “alien” impediment to DNA replication. To further develop this system as a versatile tool, we describe a set of reagents and a detailed protocol that can be used to engineer Tus-Ter barriers into any locus in the budding yeast genome. Because the Tus-Ter complex is a bipartite system with intrinsic DNA replication-blocking activity, the reagents and protocols developed and validated in yeast could also be optimized to engineer site-specific replication fork barriers into other eukaryotic cell types.

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Acknowledgments

Work in the authors’ laboratory is funded by the Danish National Research Foundation (DNRF115), The European Research Council, The Novo Nordisk Foundation, and The Nordea Foundation.

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Authors and Affiliations

  1. The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200, Copenhagen, Denmark

    Nicolai B. Larsen, Ian D. Hickson & Hocine W. Mankouri

Authors
  1. Nicolai B. Larsen
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  2. Ian D. Hickson
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  3. Hocine W. Mankouri
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Corresponding author

Correspondence to Hocine W. Mankouri .

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Editors and Affiliations

  1. Dipartimento di Bioscienze, Università degli Studi di Milano, Milano, Milano, Italy

    Marco Muzi-Falconi

  2. Donnelly Centre and Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada

    Grant W Brown

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Larsen, N.B., Hickson, I.D., Mankouri, H.W. (2018). A Molecular Toolbox to Engineer Site-Specific DNA Replication Perturbation. In: Muzi-Falconi, M., Brown, G. (eds) Genome Instability. Methods in Molecular Biology, vol 1672. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7306-4_20

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  • DOI: https://doi.org/10.1007/978-1-4939-7306-4_20

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7305-7

  • Online ISBN: 978-1-4939-7306-4

  • eBook Packages: Springer Protocols

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