Skip to main content
SpringerLink
Log in
Book cover

Cell Cycle Control pp 167–180Cite as

Mapping Origins of DNA Replication in Eukaryotes

  • Protocol

  • 2360 Accesses

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 296))

Abstract

Methods are described here to map an origin of replication in eukaryotes. Replicating DNA is enriched by BND cellulose column chromatography and by λ-exonuclease digestion; this approach has largely superceded enrichment by BrdU incorporation. The general area in which replication begins can be deciphered by neutral/neutral 2D gel electrophoresis: a restriction fragment containing the replication bubble will form a bubble arc on these gels. A more sensitive method employs PCR analysis of nascent strands that are size-fractionated. Once the general area containing the origin of bidirectional replication has been mapped, a finer level of resolution can be obtained by replication initiation point (RIP) mapping, in which start sites of DNA synthesis are identified at the nucleotide level.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Stinchcomb, D. T., Struhl, K., and Davis, R. W. (1979) Isolation and characterisation of a yeast chromosomal replicator. Nature 282, 39–43.

    Article  PubMed  CAS  Google Scholar 

  2. Brewer, B. J. and Fangman, W. L. (1987) The localization of replication origins on ARS plasmids in S. cerevisiae. Cell 51, 463–471.

    CAS  Google Scholar 

  3. Huberman, J. A., Spotila, L. D., Nawotka, K. A., El-Assouli, S. M., and Davis, L. R. (1987) The in vivo replication origin of the yeast 2 µm plasmid. Cell 51, 463–471.

    Article  Google Scholar 

  4. Newlon, C. S. and Theis, J. F. (1993) The structure and function of yeast ARS elements. Curr. Opin. Genet. Dev. 3, 752–758.

    Article  PubMed  CAS  Google Scholar 

  5. Bell, S. P. and Stillman, B. (1992) ATP-dependent recognition of eukaryotic origins of replication by a multiprotein complex. Nature 357, 128–134.

    Article  PubMed  CAS  Google Scholar 

  6. Marahrens, Y. and Stillman, B. (1992) A yeast chromosomal origin of DNA replication defined by multiple functional elements. Science 255, 817–823.

    Article  PubMed  CAS  Google Scholar 

  7. Bell, S. P. and Dutta, A. (2002) DNA replication in eukaryotic cells. Annu. Rev. Biochem. 71, 333–374.

    Article  PubMed  CAS  Google Scholar 

  8. Bielinsky, A.-K. and Gerbi, S. A. (1998) Discrete start sites for DNA synthesis in the yeast ARS1 origin. Science 279, 95–98.

    Article  PubMed  CAS  Google Scholar 

  9. Bielinsky, A.-K. and Gerbi, S. A. (1999) Chromosomal ARS1 has a single leading strand start site. Mol. Cell 3, 477–486.

    Article  PubMed  CAS  Google Scholar 

  10. Newlon, C. S., Collins I., Dershowitz, A. M., Greenfeder, S. A., Ong, L. Y., and Theis, J. F. (1993) Analysis of replication origin function on chromosome III of Saccharomyces cerevisiae. Cold Spring Harbor Symp. Quant. Biol. 58, 415–423.

    CAS  Google Scholar 

  11. Harland, R. M. and Laskey, R. A. (1980) Regulated replication of DNAmicroinjected into eggs of Xenopus laevis. Cell 21, 761–771.

    Article  CAS  Google Scholar 

  12. Méchali, M. and Kearsey, S. (1984) Lack of specific sequence requirement for DNA replication in Xenopus eggs compared with high sequence specificity in yeast. Cell 38, 55–64.

    Google Scholar 

  13. Hyrien, O. and Méchali, M. (1993) Chromosomal replication initiates and terminates at random sequences but at regular intervals in the ribosomal DNA of Xenopus early embryos. EMBOJ. 12, 4511–4520.

    CAS  Google Scholar 

  14. Shinomiya, T. and Ina, S. (1991) Analysis of chromosomal replicons in early embryos of Drosophila melanogaster by two-dimensional gel electrophoresis. Nucleic Acids Res. 19, 3935–3941.

    CAS  Google Scholar 

  15. Blow, J. J., Gillespie, P. J., Francis, D., and Jackson, D.A. (2001) Replication origins in Xenopus egg extract are 5-15 kilobases apart and are activated in clusters that fire at different times. J. Cell Biol. 152, 15–25.

    CAS  Google Scholar 

  16. Hyrien, O., Maric, C., and Méchali, M. (1995) Transition in specification of embryonic metazoan DNA replication origins. Science 270, 994–997.

    Article  PubMed  CAS  Google Scholar 

  17. Sasaki, T., Sawado, T., Yamaguchi, M., and Shinomiya, T. (1999) Specification of regions of DNA replication initiation during embryogenesis in the 65-kilobase DNA pol α-dE2F locus of Drosophila melanogaster. Mol. Cell. Biol. 19, 547–555.

    CAS  Google Scholar 

  18. Gilbert, D. M. (2001) Making sense of eukaryotic DNA replication origins. Science 294, 96–100.

    Article  PubMed  CAS  Google Scholar 

  19. Lunyak, V. V., Ezrokhi, M., Smith, H. S., and Gerbi, S. A. (2002) Developmental changes in the Sciara II/9A initiation zone for DNA replication. Mol. Cell. Biol. 22, 8426–8437.

    CAS  Google Scholar 

  20. Dijkwel, P. A. and Hamlin, J. L. (1995) The Chinese hamster dihydrofolate reductase origin consists of multiple potential nascent-strand start sites. Mol. Cell. Biol. 15, 3023–3031.

    PubMed  CAS  Google Scholar 

  21. Pelizon, C., Divacco, S., Falaschi, A., and Giacca, M. (1996) High-resolution mapping of the origin of DNA replication in the hamster dihydrofolate reductase gene domain by competitive PCR. Mol. Cell. Biol. 16, 5358–5364.

    PubMed  CAS  Google Scholar 

  22. Kobayashi, T., Rein, T., and DePamphilis, M. L. (1998) Identification of primary initiation sites for DNA replication in the hamster dihydrofolate reductase gene initiation zone. Mol. Cell. Biol. 18, 3266–3277.

    PubMed  CAS  Google Scholar 

  23. Gomez, M. and Antequera, F. (1999) Organization of DNA replication origins in the fission yeast genome. EMBO J. 18, 5683–5690.

    Article  PubMed  CAS  Google Scholar 

  24. Bielinsky, A.-K., Blitzblau, H., Beall, E.L., et al. (2001) Origin recognition complex binding to a metazoan origin. Curr. Biol. 11, 1–20.

    Article  Google Scholar 

  25. Abdurashidova, G., Deganuto, M., Klima, R., et al. (2000) Start site of bidirectional DNA synthesis at the human lamin B2 origin. Science 287, 2023–2026.

    Article  PubMed  CAS  Google Scholar 

  26. Gerbi, S. A., Bielinsky, A. K., Liang, C., Lunyak, V. V., and Urnov, F.D. (1999) Methods to map origins of replication in eukaryotes, in Eukaryotic DNA Replication (Cottterill, S., ed.), Oxford University Press, Oxford, pp. 1–42.

    Google Scholar 

  27. Liang, C. and Gerbi, S. A. (1994) Analysis of an origin of DNA amplification in Sciara coprophila by a novel three-dimensional gel method. Mol. Cell. Biol. 14, 1520–1559.

    CAS  Google Scholar 

  28. Vassilev, L. and Johnson, E. M. (1989). Maping intiation sites of DNA replication in vivo using polymerase chain reaction amplification of nascent strand segments. Nucleic Acids Res. 19, 7693–7705.

    Article  Google Scholar 

  29. Diviacco, S., Norio, P., Zentilin, L., et al. (1992) A novel procedure for quantitative polymerase chain reaction by coamplification of competitive templates. Gene 122, 313–320.

    Article  PubMed  CAS  Google Scholar 

  30. Hay, R. and DePamphilis, M. L. (1982) Initiation of SV40 DNA replication in vivo: location and structure of 5′ ends of DNA synthesized in the ORI region. Cell 28, 767–779.

    CAS  Google Scholar 

Download references

Authors
  1. Susan A. Gerbi
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. MRC Radiation and Genome Stability Unit, Harwell, Didcot, UK

    Tim Humphrey

  2. School of Pharmacy, University of Reading Whiteknights, Reading, UK

    Gavin Brooks

Rights and permissions

Reprints and permissions

Copyright information

© 2005 Humana Press Inc.

About this protocol

Cite this protocol

Gerbi, S.A. (2005). Mapping Origins of DNA Replication in Eukaryotes. In: Humphrey, T., Brooks, G. (eds) Cell Cycle Control. Methods in Molecular Biology™, vol 296. Humana Press. https://doi.org/10.1385/1-59259-857-9:167

Download citation

  • DOI: https://doi.org/10.1385/1-59259-857-9:167

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-144-8

  • Online ISBN: 978-1-59259-857-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation