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Homologous Recombination pp 355–363Cite as

Super-Resolution Imaging of Homologous Recombination Repair at Collapsed Replication Forks

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

Abstract

Single-molecule super-resolution microscopy (SRM) combines single-molecule detection with spatial resolutions tenfold improved over conventional confocal microscopy. These two key advantages make it possible to visualize individual DNA replication and damage events within the cellular context of fixed cells. This in turn engenders the ability to decipher variations between individual replicative and damage species within a single nucleus, elucidating different subpopulations of stress and repair events. Here, we describe the protocol for combining SRM with novel labeling and damage assays to characterize DNA double-strand break (DSB) induction at stressed replication forks (RFs) and subsequent repair by homologous recombination (HR). These assays enable spatiotemporal mapping of DNA damage response and repair proteins to establish their in vivo function and interactions, as well as detailed characterization of specific dysfunctions in HR caused by drugs or mutations of interest.

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Acknowledgments

D.R.W. would like to acknowledge support from a Bruce Stone Fellowship from the La Trobe Institute for Molecular Science and funding via the Bendigo Tertiary Education Anniversary Foundation. Research in the Rothenberg lab is supported by funds from the NIH R01 GM108119, American Cancer Society (ACS: 130304-RSG-16-241-01-DMC), the V Foundation for Cancer Research (D2018-020), and Fondation Leducq (17CVD02).

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Authors and Affiliations

  1. Department of Pharmacy and Biomedical Sciences, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, VIC, Australia

    Donna R. Whelan

  2. Department of Biochemistry and Molecular Pharmacology, Perlmutter Cancer Center, New York University School of Medicine, New York, NY, USA

    Donna R. Whelan & Eli Rothenberg

Authors
  1. Donna R. Whelan
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  2. Eli Rothenberg
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Corresponding author

Correspondence to Donna R. Whelan .

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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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Whelan, D.R., Rothenberg, E. (2021). Super-Resolution Imaging of Homologous Recombination Repair at Collapsed 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_24

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  • DOI: https://doi.org/10.1007/978-1-0716-0644-5_24

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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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