Abstract
How cells duplicate their chromosomes is a key determinant of cell identity and genome stability. DNA replication can initiate from more than 100,000 sites distributed along mammalian chromosomes, yet a given cell uses only a subset of these origins due to inefficient origin activation and regulation by developmental or environmental cues. An impractical consequence of cell-to-cell variations in origin firing is that population-based techniques do not accurately describe how chromosomes are replicated in single cells. DNA combing is a biophysical DNA fiber stretching method which permits visualization of ongoing DNA synthesis along Mb-sized single-DNA molecules purified from cells that were previously pulse-labeled with thymidine analogues. This allows quantitative measurements of several salient features of chromosome replication dynamics, such as fork velocity, fork asymmetry, inter-origin distances, and global instant fork density. In this chapter we describe how to obtain this information from asynchronous cultures of mammalian cells.
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Acknowledgments
The Montpellier DNA combing facility is part of BioCampus Montpellier (www.biocampus.cnrs.fr) and has financial support from Cancéropôle Grand Sud-Ouest, SIRIC Montpellier, Région Languedoc-Roussillon and BioCampus Montpellier. ES acknowledges CNRS, Institut National du Cancer (INCa), Fondation pour la Recherche Médicale (FRM), and Association pour la Recherche sur le Cancer (ARC) for funding. MB has a Ph.D. fellowship from the Ministry of Research (MENRT).
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Bialic, M., Coulon, V., Drac, M., Gostan, T., Schwob, E. (2015). Analyzing the Dynamics of DNA Replication in Mammalian Cells Using DNA Combing. In: Vengrova, S., Dalgaard, J. (eds) DNA Replication. Methods in Molecular Biology, vol 1300. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2596-4_4
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DOI: https://doi.org/10.1007/978-1-4939-2596-4_4
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