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Measurement of Homologous Recombination at Stalled Mammalian Replication Forks

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Homologous Recombination

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2153))

Abstract

Site-specific replication fork barriers (RFBs) have proven valuable tools for studying mechanisms of repair at sites of replication fork stalling in prokaryotes and yeasts. We adapted the Escherichia coli Tus-Ter RFB for use in mammalian cells and used it to trigger site-specific replication fork stalling and homologous recombination (HR) at a defined chromosomal locus in mammalian cells. By comparing HR responses induced at the Tus-Ter RFB with those induced by a site-specific double-strand break (DSB), we have begun to uncover how the mechanisms of mammalian stalled fork repair differ from those underlying the repair of a replication-independent DSB. Here, we outline how to transiently express the Tus protein in mES cells, how to use flow cytometry to score conservative and aberrant repair outcomes, and how to quantify distinct repair outcomes in response to replication fork stalling at the inducible Tus-Ter chromosomal RFB.

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Authors and Affiliations

  1. Department of Medicine, Division of Hematology-Oncology, Cancer Research Institute, Harvard Medical School, Boston, MA, USA

    Nicholas A. Willis & Ralph Scully

  2. Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

    Nicholas A. Willis & Ralph Scully

Authors
  1. Nicholas A. Willis
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  2. Ralph Scully
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Corresponding author

Correspondence to Ralph Scully .

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Editors and Affiliations

  1. Centro Andaluz de Biologı´a Molecular, y Medicina Regenerativa, CABIMER, Universidad de Sevilla, Sevilla, Spain

    Andrés Aguilera

  2. Institut Curie, PSL Research University, CNRS, UMR3348, F-91405, Orsay, France

    Aura Carreira

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Willis, N.A., Scully, R. (2021). Measurement of Homologous Recombination at Stalled Mammalian Replication Forks. In: Aguilera, A., Carreira, A. (eds) Homologous Recombination. Methods in Molecular Biology, vol 2153. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0644-5_23

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  • DOI: https://doi.org/10.1007/978-1-0716-0644-5_23

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0643-8

  • Online ISBN: 978-1-0716-0644-5

  • eBook Packages: Springer Protocols

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