Summary
The Xenopus egg extract has become the gold standard for in vitro studies of metazoan DNA replication. We have used this system to study the mechanisms that ensure rapid and complete DNA replication despite random initiation during Xenopus early development. To this end we adapted the DNA combing technique to investigate the distribution of replication bubbles along single DNA molecules. DNA replicating in egg extracts is labelled by addition of digoxigenin-11-dUTP and/or biotin-16-dUTP at precise times. These two dTTP analogues are efficiently incorporated into DNA during replication in the extract. After DNA purification and combing the DNA is visualized with appropriate fluorescent antibody/streptavidin molecules. Replicated DNA appears as green or red tracts whose pattern reveals how each molecule was replicated, allowing to follow the dynamics of DNA replication through S phase. We describe (a) the preparation and use of egg extracts and demembranated sperm chromatin templates; (b) a simple method for preparing silanized glass coverslips suitable for DNA combing and fluorescence detection; (c) two alternative replicative DNA labelling schemes and their respective advantages; and (d) a protocol for combining replicative labelling with detection of specific DNA sequences by fluorescent in situ hybridization (FISH).Although most observations made in Xenopus egg extracts are applicable to other eukaryotes, there are differences in cell-cycle regulation between mammalian somatic cells and embryonic amphibian cells, which led to the development of human cell-free systems that can initiate semi-conservative chromosomal DNA replication under cell-cycle control. We have employed the knowledge gained with Xenopus extracts to characterize DNA replication intermediates generated in human cell-free systems using DNA combing. We describe here (a) the preparation and use of human cell-free extracts and initiation-competent template nuclei for DNA combing studies; (b) an optimized labelling scheme for DNA replication intermediates by molecular combing and fluorescence microscopy.
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References
DePamphilis, M. L. (1997) DNA replication, Methods 13, 209–210
Hyrien, O., Marheineke, K., and Goldar, A. (2003) Paradoxes of eukaryotic DNA replication: MCM proteins and the random completion problem, BioEssays 25, 116–125
Hyrien, O., Maric, C., and M×échali, M. (1995) Transition in specification of embryonic metazoan DNA replication origins, Science 270, 994–997
Hyrien, O., and M×échali, M. (1993) Chromosomal replication initiates and terminates at random sequences but at regular intervals in the ribosomal DNA of Xenopus early embryos, EMBO J 12, 4511–4520
Hyrien, O., and M×échali, M. (1992) Plasmid replication in Xenopus eggs and egg extracts: a 2D gel electrophoretic analysis, Nucleic Acids Res 20, 1463–1469
Mahbubani, H. M., Paull, T., Elder, J. K., and Blow, J. J. (1992) DNA replication initiates at multiple sites on plasmid DNA in Xenopus egg extracts, Nucleic Acids Res 20, 1457–1462
Blumenthal, A. B., Kriegstein, H. J., and Hogness, D. S. (1974) The units of DNA replication in Drosophila melanogaster chromosomes, Cold Spring Harb Symp Quant Biol 38, 205–223
Lucas, I., Chevrier-Miller, M., Sogo, J. M., and Hyrien, O. (2000) Mechanisms ensuring rapid and complete DNA replication despite random initiation in Xenopus early embryos, J Mol Biol 296, 769–786
Huberman, J. A., and Riggs, A. D. (1968) On the mechanism of DNA replication in mammalian chromosomes, J Mol Biol 32, 327–341
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, 2096–2098
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, 1518–1523
Herrick, J., Stanislawski, P., Hyrien, O., and Bensimon, A. (2000) Replication fork density increases during DNA synthesis in X. laevis egg extracts, J Mol Biol 300, 1133–1142
Marheineke, K., and Hyrien, O. (2001) Aphidicolin triggers a block to replication origin firing in Xenopus egg extracts, J Biol Chem 276, 17092–17100
Marheineke, K., and Hyrien, O. (2004) Control of replication origin density and firing time in Xenopus egg extracts: role of a caffeine-sensitive, ATR-dependent checkpoint, J Biol Chem 279, 28071–28081.
Marheineke, K., Hyrien, O., and Krude, T. (2005) Visualization of bidirectional initiation of chromosomal DNA replication in a human cell free system, Nucleic Acids Res 33, 6931–6941
Krude, T. (2006) Initiation of chromosomal DNA replication in mammalian cell-free systems, Cell Cycle 5, 2115–2122
. Krude, T., Christov, C., Hyrien, O., and Marheineke, K. (2008) Y RNA Functions at the initiation step of mammalian chromosomal DNA replication, submitted
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, 1433–1442
Lemaitre, J. M., Danis, E., Pasero, P., Vassetzky, Y., and Mechali, M. (2005) Mitotic remodeling of the replicon and chromosome structure, Cell 123, 787–801
Pillaire, M. J., Betous, R., Conti, C., Czaplicki, J., Pasero, P., Bensimon, A., Cazaux, C., and Hoffmann, J. S. (2007) Upregulation of error-prone DNA polymerases beta and kappa slows down fork progression without activating the replication checkpoint, Cell Cycle 6, 471–477
Conti, C., Sacca, B., Herrick, J., Lalou, C., Pommier, Y., and Bensimon, A. (2007) Replication fork velocities at adjacent replication origins are coordinately modified during DNA replication in human cells, Mol Biol Cell 18, 3059–3067
Pasero, P., Bensimon, A., and Schwob, E. (2002) Single-molecule analysis reveals clustering and epigenetic regulation of replication origins at the yeast rDNA locus, Genes Dev 16, 2479–2484
Anglana, M., Apiou, F., Bensimon, A., and Debatisse, M. (2003) Dynamics of DNA replication in mammalian somatic cells: nucleotide pool modulates origin choice and interorigin spacing, Cell 114, 385–394
Lebofsky, R., and Bensimon, A. (2005) DNA replication origin plasticity and perturbed fork progression in human inverted repeats, Mol Cell Biol 25, 6789–6797
. Lebofsky, R., Heilig, R., Sonnleitner, M., Weissenbach, J., and Bensimon, A. (2006) DNA replication origin interference increases the spacing between initiation events in human cells, Mol Biol Cell 17, 5337–5345
Allemand, J. F., Bensimon, D., Jullien, L., Bensimon, A., and Croquette, V. (1997) pH-dependent specific binding and combing of DNA, Biophys J 73, 2064–2070
.Labit, H., Goldar, A., Guilbaud, G., Douarche, C., Hyrien, O., and Marheineke, K. (2008) An optimized easy method for preparing silanized surfaces for FISH and replication mapping on combed DNA fibers, Biotechniques, in press
Gurdon, J. B. (1976) Injected nuclei in frog oocytes: fate, enlargement, and chromatin dispersal, J Embryol Exp Morphol 36, 523–540
Murray, A. W. (1991) Cell cycle extracts, Methods Cell Biol 36, 581–605
Blow, J. J., and Laskey, R. A. (1986) Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs, Cell 47, 577–587
Szuts, D., and Krude, T. (2004) Cell cycle arrest at the initiation step of human chromosomal DNA replication causes DNA damage, J Cell Sci 117, 4897–4908
Krude, T. (1999) Mimosine arrests proliferating human cells before onset of DNA replication in a dose-dependent manner, Exp Cell Res 247, 148–159
Krude, T., Jackman, M., Pines, J., and Laskey, R. A. (1997) Cyclin/Cdk-dependent initiation of DNA replication in a human cell-free system, Cell 88, 109–119
Li, J. J., and Kelly, T. J. (1984) Simian virus 40 DNA replication in vitro, Proc Natl Acad Sci U S A 81, 6973–6977
Krude, T. (2000) Initiation of human DNA replication in vitro using nuclei from cells arrested at an initiation-competent state, J Biol Chem 275, 13699–13707
Keller, C., Hyrien, O., Knippers, R., and Krude, T.(2002) Site-specific and temporally controlled initiation of DNA replication in a human cell-free system, Nucleic Acids Res. 30, 2114–2123
Brzoska, J., Shahidzadeh, N., and Rondelez, F. (1992) Evidence of a transition temperature for the optimum deposition of grafted monolayer coatings, Nature 360, 719–721
Dong, J., Wang, A., Simon Ng, K., and Mao, G.(2006) Self-assembly of octadecyltrichlorosilane monolayers on silicon-based substrates by chemical vapour deposition, Thin Solid Films 515, 2116–2122
Stoeber, K., Mills, A. D., Kubota, Y., Krude, T., Romanowski, P., Marheineke, K., Laskey, R. A., and Williams, G. H. (1998) Cdc6 protein causes premature entry into S phase in a mammalian cell- free system, EMBO J 17, 7219–7229
Angst, D., and Simmons, G. (1991) Moisture absorption characteristics of organosiloxane self-assembled monolayers, Langmuir 7, 2236–2242
Acknowledgements
We thank J-F Allemand (ENS, Paris) for many helpful discussions and for providing us a motorized device of his design to comb DNA; and B. Berge, Z. Gueroui, and C. Place (ENS-Lyon) for providing us silanized coverslips prepared by gas-phase silanization. The T.K. lab is supported by Cancer Research UK, the O.H. lab by the Association pour la Recherche sur le Cancer, the Ligue Nationale contre le Cancer (Comit×é de Paris), the Agence Nationale pour la Recherche and the Fondation pour la RechercheM×édicale, the A.G. lab by the Association pour la Recherche sur le Cancer, the Agence Nationale pour la Recherche and the Commissariat ×° l'Energie Atomique.
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Marheineke, K., Goldar, A., Krude, T., Hyrien, O. (2009). Use of DNA Combing to Study DNA Replicationin Xenopus and Human Cell-Free Systems. 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_33
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DOI: https://doi.org/10.1007/978-1-60327-815-7_33
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