Summary
Plasticity is an inherent feature of chromosomal DNA replication in eukaryotes. Potential origins of DNA replication are made in excess, but are used (fired) in a partly stochastic, partly programmed manner throughout the S phase of the cell cycle. Since most origins have a firing efficiency below 50%, population-based analysis methods yield a cumulative picture of origin activity (obtained by accretion) that does not accurately describe how chromosomes are replicated in single cells. DNA combing is a method that allows the alignment on silanized glass coverslips, at high density and with uniform stretching, of single DNA molecules in the Mb range. If this DNA is isolated from cells that have been labelled with halogenated nucleotides (BrdU, CldU, IdU), it is possible to determine the density and position of replication origins as well as the rate and symmetry of fork progression, quantitatively and on single DNA molecules. This chapter will successively describe (a) the preparation of silanized coverslips, (b) the incorporation of halogenated nucleotides in newly synthesized DNA in yeast and mammalian cell lines, (c) the preparation and combing of genomic DNA, and finally (d) the acquisition and analysis of single-molecule images to extract salient features of replication dynamics.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Zlatanova, J., and van Holde, K. (2006) Single-molecule biology: what is it and how does it work? Mol Cell24, 317–29.
Bancaud, A., Conde e Silva, N., Barbi, M., Wagner, G., Allemand, J. F., Mozziconacci, J., Lavelle, C., Croquette, V., Victor, J. M., Prunell, A., and Viovy, J. L. (2006) Structural plasticity of single chromatin fibers revealed by torsional manipulation. Nat Struct Mol Biol 13, 444–50.
Lia, G., Praly, E., Ferreira, H., Stockdale, C., Tse-Dinh, Y. C., Dunlap, D., Croquette, V., Bensimon, D., and Owen-Hughes, T. (2006) Direct observation of DNA distortion by the RSC complex. Mol Cell 21, 417–25.
Strick, T. R., Croquette, V., and Bensimon, D. (2000) Single-molecule analysis of DNA uncoiling by a type II topoisomerase. Nature 404, 901–4.
Michalet, X., Pinaud, F. F., Bentolila, L. A., Tsay, J. M., Doose, S., Li, J. J., Sundaresan, G., Wu, A. M., Gambhir, S. S., and Weiss, S. (2005) Quantum dots for live cells, in vivo imaging, and diagnostics. Science 307, 538–44.
Elowitz, M. B., Levine, A. J., Siggia, E. D., and Swain, P. S. (2002) Stochastic gene expression in a single cell. Science 297, 1183–6.
Raser, J. M., and O’Shea, E. K. (2005) Noise in gene expression: origins, consequences, and control. Science 309, 2010–3.
Patel, P. K., Arcangioli, B., Baker, S. P., Bensimon, A., and Rhind, N. (2006) DNA replication origins fire stochastically in fission yeast. Mol Biol Cell 17, 308–16.
Di Talia, S., Skotheim, J. M., Bean, J. M., Siggia, E. D., and Cross, F. R. (2007) The effects of molecular noise and size control on variability in the budding yeast cell cycle. Nature 448, 947–51.
Bensimon, A., Simon, A., Chiffaudel, A., Croquette, V., Heslot, F., and Bensimon, D. (1994) Alignment and sensitive detection of DNA by a moving interface. Science 265, 2096–8.
Michalet, X., Ekong, R., Fougerousse, F., Rousseaux, S., Schurra, C., Hornigold, N., van Slegtenhorst, M., Wolfe, J., Povey, S., Beckmann, J. S., and Bensimon, A. (1997) Dynamic molecular combing: stretching the whole human genome for high- resolution studies. Science 277, 1518–23.
Allemand, J. F., Bensimon, D., Jullien, L., Bensimon, A., and Croquette, V. (1997) pH-dependent specific binding and combing of DNA. Biophys J 73, 2064–70.
Jung, G. Y., Li, Z., Wu, W., Chen, Y., Olynick, D. L., Wang, S. Y., Tong, W. M., and Williams, R. S. (2005) Vapor-phase self-assembled monolayer for improved mold release in nanoimprint lithography. Langmuir 21, 1158–61.
Bunker, B. C., Carpick, R. W., Assink, R. A., Thomas, M. L., Hankins, M. G., Voigt, J. A., Sipola, D., de Boer, M. P., and Gulley, G. L.(2000) The Impact of Solution Agglomeration on the Deposition of Self-Assembled Monolayers. Langmuir 16, 7742–7751.
Dolbeare, F. (1996) Bromodeoxyuridine: a diagnostic tool in biology and medicine, Part III. Proliferation in normal, injured and diseased tissue, growth factors, differentiation, DNA replication sites and in situ hybridization. Histochem J 28, 531–75.
Lengronne, A., Pasero, P., Bensimon, A., and Schwob, E. (2001) Monitoring S phase progression globally and locally using BrdU incorporation in TK(+) yeast strains. Nucleic Acids Res 29, 1433–42.
Viggiani, C. J., and Aparicio, O. M. (2006) New vectors for simplified construction of BrdU-Incorporating strains of Saccharomyces cerevisiae. Yeast 23, 1045–51.
Sivakumar, S., Porter-Goff, M., Patel, P. K., Benoit, K., and Rhind, N. (2004) In vivo labelling of fission yeast DNA with thymidine and thymidine analogs. Methods 33, 213–9.
Acknowledgements
We thank Aaron Bensimon (Genomic Vision) for providing initial access to the methodology and the Pasero lab (IGH Montpellier) for continuous scientific and technical exchanges. The Montpellier DNA combing facility was launched with support from the Cancéropôle Grand Sud-Ouest. ES acknowledges CNRS, Institut National du Cancer (INCa PL110), and Association pour la Recherche sur le Cancer (ARC SL3149) for funding. CR was supported by PhD fellowships from MENRT and ARC, VC by post-doctoral fellowships from CNRS and INCa.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Schwob, E. et al. (2009). Use of DNA Combing for Studying DNA Replication In Vivo in Yeast and Mammalian Cells. In: Vengrova, S., Dalgaard, J. (eds) DNA Replication. Methods in Molecular Biology, vol 521. Humana Press. https://doi.org/10.1007/978-1-60327-815-7_36
Download citation
DOI: https://doi.org/10.1007/978-1-60327-815-7_36
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60327-814-0
Online ISBN: 978-1-60327-815-7
eBook Packages: Springer Protocols