Abstract
The ability of cells to copy their DNA allows them to transmit their genetic information to their progeny. In such, this central biological process preserves the instructions that direct the entire development of a cell. Earlier biochemical analysis in vitro and genetic analysis in yeast laid the basis of our understanding of the highly conserved mechanism of DNA replication. Recent advances on labeling and live-cell microscopy permit now the dissection of this fundamental process in vivo within the context of intact cells. In this chapter, we describe in detail how to perform multiple DNA replication labeling and detection allowing high spatial resolution imaging, as well as how to follow DNA replication in living cells allowing high temporal resolution imaging.
Marius Reinhart and Corella S. Casas-Delucchi have contributed equally to this work.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Berezney R, Dubey DD, Huberman JA (2000) Heterogeneity of eukaryotic replicons, replicon clusters, and replication foci. Chromosoma 108:471–484
Nakamura H, Morita T, Sato C (1986) Structural organizations of replicon domains during DNA synthetic phase in the mammalian nucleus. Exp Cell Res 165:291–297
Nakayasu H, Berezney R (1989) Mapping replicational sites in the eucaryotic cell nucleus. J Cell Biol 108:1–11
O’Keefe RT, Henderson SC, Spector DL (1992) Dynamic organization of DNA replication in mammalian cell nuclei: spatially and temporally defined replication of chromosome-specific alpha-satellite DNA sequences. J Cell Biol 116:1095–1110
Huberman JA, Riggs AD (1966) Autoradiography of chromosomal DNA fibers from Chinese hamster cells. Proc Natl Acad Sci USA 55:599–606
Huberman JA, Tsai A, Deich RA (1973) DNA replication sites within nuclei of mammalian cells. Nature 241:32–36
Gratzner HG (1982) Monoclonal antibody to 5-bromo- and 5-iododeoxyuridine: a new reagent for detection of DNA replication. Science 218:474–475
Bravo R, Macdonald-Bravo H (1985) Changes in the nuclear distribution of cyclin (PCNA) but not its synthesis depend on DNA replication. EMBO J 4:655–661
Cardoso MC, Leonhardt H, Nadal-Ginard B (1993) Reversal of terminal differentiation and control of DNA replication: cyclin A and Cdk2 specifically localize at subnuclear sites of DNA replication. Cell 74:979–992
Aten JA, Bakker PJ, Stap J, Boschman GA, Veenhof CH (1992) DNA double labelling with IdUrd and CldUrd for spatial and temporal analysis of cell proliferation and DNA replication. Histochem J 24:251–259
Manders E, Stap J, Brakenhoff G, Van Driel R, Aten J (1992) Dynamics of three-dimensional replication patterns during the S-phase, analysed by double labelling of DNA and confocal microscopy. J Cell Sci 103:857–862
Schermelleh L, Solovei I, Zink D, Cremer T (2001) Two-color fluorescence labeling of early and mid-to-late replicating chromatin in living cells. Chromosome Res 9:77–80
Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 263:802–805
Cardoso MC, Joseph C, Rahn HP, Reusch R, Nadal-Ginard B, Leonhardt H (1997) Mapping and use of a sequence that targets DNA ligase I to sites of DNA replication in vivo. J Cell Biol 139:579–587
Leonhardt H, Rahn H, Weinzierl P, Sporbert A, Cremer T, Zink D, Cardoso M (2000) Dynamics of DNA replication factories in living cells. J Cell Biol 149:271–280
Sporbert A, Gahl A, Ankerhold R, Leonhardt H, Cardoso MC (2002) DNA polymerase clamp shows little turnover at established replication sites but sequential de novo assembly at adjacent origin clusters. Mol Cell 10:1355–1365
Chagin VO, Stear JH, Cardoso MC (2010) Organization of DNA Replication. Cold Spring Harb Perspect Biol 2
Casas-Delucchi CS, Brero A, Rahn H-P, Solovei I, Wutz A, Cremer T, Leonhardt H, Cardoso MC (2011) Histone acetylation controls the inactive X chromosome replication dynamics. Nat commun 2:222
Heisenberg W (1927) Über den anschaulichen Inhalt der quantentheoretischen Kinematik und Mechanik. Zeitschrift für Physik 43:172–198
Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2:905–909
Sporbert A, Domaing P, Leonhardt H, Cardoso MC (2005) PCNA acts as a stationary loading platform for transiently interacting Okazaki fragment maturation proteins. Nucleic Acids Res 33:3521–3528
Bullejos M, Burgos M, Jimenez R, Sanchez A (1996) Distribution of sister-chromatid exchanges in different types of chromatin in the X-chromosome of Microtus cabrerae. Experientia 52:511–515
Lindhout BI, Fransz P, Tessadori F, Meckel T, Hooykaas PJJ, van der Zaal BJ (2007) Live cell imaging of repetitive DNA sequences via GFP-tagged polydactyl zinc finger proteins. Nucleic Acids Res 35:e107
Casas-Delucchi CS, van Bemmel JG, Haase S, Herce HD, Nowak D, Meilinger D, Stear JH, Leonhardt H, Cardoso MC (2012) Histone hypoacetylation is required to maintain late replication timing of constitutive heterochromatin. Nucleic Acids Res 40:159–169
Weidtkamp-Peters S, Rahn H-P, Cardoso MC, Hemmerich P (2006) Replication of centromeric heterochromatin in mouse fibroblasts takes place in early, middle, and late S phase. Histochem Cell Biol 125:91–102
Cardoso MC, Leonhardt H (1995). Immunofluorescence techniques in cell cycle studies. In: Pagano, M (ed) Cell cycle: materials and methods. Heidelberg, Springer-Verlag, pp 15–28
Acknowledgements
We thank Juan Alberto Marchal (University of Jaen, Spain) for the Microtus cabrerae fibroblasts and all present and past members of the laboratory for their contributions over the years. The laboratory of M. Cristina Cardoso is supported by grants of the German Research Foundation (DFG) and the Federal Ministry of Education and Research (BMBF).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Reinhart, M., Casas-Delucchi, C.S., Cardoso, M.C. (2013). Spatiotemporal Visualization of DNA Replication Dynamics. In: Shav-Tal, Y. (eds) Imaging Gene Expression. Methods in Molecular Biology, vol 1042. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-526-2_15
Download citation
DOI: https://doi.org/10.1007/978-1-62703-526-2_15
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-525-5
Online ISBN: 978-1-62703-526-2
eBook Packages: Springer Protocols