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Dynamic Architecture of Eukaryotic DNA Replication Forks In Vivo, Visualized by Electron Microscopy

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Book cover Genome Instability

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

Abstract

The DNA replication process can be heavily perturbed by several different conditions of genotoxic stress, particularly relevant for cancer onset and therapy. The combination of psoralen crosslinking and electron microscopy has proven instrumental to reveal the fine architecture of in vivo DNA replication intermediates and to uncover their remodeling upon specific conditions of genotoxic stress. The replication structures are stabilized in vivo (by psoralen crosslinking) prior to extraction and enrichment procedures, allowing their visualization at the transmission electron microscope. This chapter outlines the procedures required to visualize and interpret in vivo replication intermediates of eukaryotic genomic DNA, and includes an improved method for enrichment of replication intermediates, compared to previously used BND-cellulose columns.

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Acknowledgments

We are grateful to José M. Sogo for his crucial support while learning all technicalities of this approach. We wish to thank the whole team at the ZMB (Center for Microscopy and Image Analysis of the University Zurich) for consistently excellent technical assistance, while running our EM experiments. We are grateful to Arnab Ray Chaudhuri, Yoshitami Hashimoto, Fabio Puddu, and Vincenzo Costanzo for their assistance in optimizing this EM approach on Xenopus egg extracts. We also wish to thank Petr Cejka for suggesting the use of QIAGEN columns as possible valuable alternative to BND cellulose for the enrichment of a subpopulation of DNA molecules based on differential ssDNA content. We are also grateful to Sebastian Ursich for his recent efforts optimizing this approach and for careful reading of the manuscript. Work in the Lopes lab is currently financed by the SNF grant 31003A_169959, the ERC Consolidator grant 617102 (ReStreCa) and the Swiss Cancer League grant KFS-3967-08-2016.

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Authors and Affiliations

  1. University of Zurich, Institute of Molecular Cancer Research, Zurich, Switzerland

    Ralph Zellweger & Massimo Lopes

Authors
  1. Ralph Zellweger
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  2. Massimo Lopes
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Correspondence to Massimo Lopes .

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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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Zellweger, R., Lopes, M. (2018). Dynamic Architecture of Eukaryotic DNA Replication Forks In Vivo, Visualized by Electron Microscopy. 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_19

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  • DOI: https://doi.org/10.1007/978-1-4939-7306-4_19

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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

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