Abstract
The detailed understanding of the DNA replication process requires structural insight. The combination of psoralen cross-linking and electron microscopy has been extensively exploited to reveal the fine architecture of in vivo DNA replication intermediates. This approach proved instrumental to uncover the basic mechanisms of DNA duplication, as well as the perturbation of this process by various forms of replication stress. The replication structures are stabilized in vivo (by psoralen cross-linking) 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 genomic DNA, extracted from budding yeast, Xenopus egg extracts, or cultured mammalian cells.
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Acknowledgments
We are grateful to Dr. José M. Sogo for the patient coaching while learning these techniques. We wish to thank Gery Barmettler, Bruno Guhl, Andres Kaech, Urs Ziegler, and the whole team at the ZMB (Center for Microscopy and Image Analysis of the University Zurich) for excellent technical assistance running the EM experiments. We are also grateful to Yoshitami Hashimoto, Fabio Puddu, and Vincenzo Costanzo (Clare Hall, Cancer Research UK) for their assistance in optimizing this EM approach on Xenopus egg extracts.
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Neelsen, K.J., Chaudhuri, A.R., Follonier, C., Herrador, R., Lopes, M. (2014). Visualization and Interpretation of Eukaryotic DNA Replication Intermediates In Vivo by Electron Microscopy. In: Stockert, J., Espada, J., Blázquez-Castro, A. (eds) Functional Analysis of DNA and Chromatin. Methods in Molecular Biology, vol 1094. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-706-8_15
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DOI: https://doi.org/10.1007/978-1-62703-706-8_15
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