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Studying Single-Stranded DNA Gaps at Replication Intermediates by Electron Microscopy

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DNA Damage Responses

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2444))

Abstract

Single-stranded DNA gaps are frequent structures that accumulate on newly synthesized DNA under conditions of replication stress. The identification of these single-stranded DNA gaps has been instrumental to uncover the mechanisms that allow the DNA replication machinery to skip intrinsic replication obstacles or DNA lesions. DNA fiber assays provide an essential tool for detecting perturbations in DNA replication fork dynamics genome-wide at single molecule resolution along with identifying the presence of single-stranded gaps when used in combination with S1 nuclease. However, electron microscopy is the only technique allowing the actual visualization and localization of single-stranded DNA gaps on replication forks. This chapter provides a detailed method for visualizing single-stranded DNA gaps at the replication fork by electron microscopy including psoralen cross-linking of cultured mammalian cells, extraction of genomic DNA, and finally enrichment of replication intermediates followed by spreading and platinum rotary shadowing of the DNA onto grids. Discussion on identification and analysis of these gaps as well as on the advantages and disadvantages of electron microscopy relative to the DNA fiber technique is also included.

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Acknowledgments

We thank Alice Meroni and Annabel Quinet for their help with the preparation of the figures and for their comments on the manuscript. This work was supported by the NCI under grant numbers R01CA237263 and R01CA248526 and by the DOD BCRP Expansion Award BC191374 to A.V. This research was supported by the Alvin J. Siteman Cancer Center Siteman Investment Program (supported by The Foundation for Barnes-Jewish Hospital, Cancer Frontier Fund) and the Barnard Foundation to A.V. Figures were created with BioRender.com.

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Authors and Affiliations

  1. Division of Oncology, Department of Medicine, Washington University in St. Louis, St. Louis, MO, USA

    Jessica Jackson & Alessandro Vindigni

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  1. Jessica Jackson
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  2. Alessandro Vindigni
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Correspondence to Alessandro Vindigni .

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  1. Department of Pathology and Immunology, Washington University in St. Louis School of Medicine, St. Louis, MO, USA

    Nima Mosammaparast

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Jackson, J., Vindigni, A. (2022). Studying Single-Stranded DNA Gaps at Replication Intermediates by Electron Microscopy. In: Mosammaparast, N. (eds) DNA Damage Responses. Methods in Molecular Biology, vol 2444. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2063-2_6

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  • DOI: https://doi.org/10.1007/978-1-0716-2063-2_6

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2062-5

  • Online ISBN: 978-1-0716-2063-2

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