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Xenopus Protocols pp 121–137Cite as

Chromosomal DNA Replication in a Soluble Cell-Free System Derived From Xenopus Eggs

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Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 322))

Abstract

Cytoplasmic egg extracts from the frog Xenopus laevis represent a powerful cell-free system to study eukaryotic chromosomal DNA replication. In the classical approach, sperm chromatin is added to unfractionated egg cytoplasm, leading to the assembly of transport-competent nuclei that undergo a single, complete round of DNA replication. The need for nuclei in this system has been circumvented. Sperm chromatin or plasmid DNA is first incubated with clarified egg cytoplasm to form chromatin-bound prereplication complexes. Subsequently, a highly concentrated nucleoplasmic extract is added that stimulates initiation from these prereplication complexes, and a single complete round of chromosomal DNA replication ensues. This review describes the preparation of the cytosolic and nucleoplasmic extracts, as well as their use in DNA replication, origin unwinding, and chromatin isolation assays.

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Author information

Authors and Affiliations

  1. Novartis Institutes for Biomedical Research, Cambridge, MA

    Antonin V. Tutter

  2. Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA

    Johannes C. Walter

Authors
  1. Antonin V. Tutter
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  2. Johannes C. Walter
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Editor information

Editors and Affiliations

  1. Ottawa Health Research Institute, Ottawa Hospital, Ottawa, Ontario, Canada

    X. Johné Liu

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© 2006 Humana Press Inc., Totowa, NJ

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Tutter, A.V., Walter, J.C. (2006). Chromosomal DNA Replication in a Soluble Cell-Free System Derived From Xenopus Eggs. In: Liu, X.J. (eds) Xenopus Protocols. Methods in Molecular Biology™, vol 322. Humana Press. https://doi.org/10.1007/978-1-59745-000-3_9

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  • DOI: https://doi.org/10.1007/978-1-59745-000-3_9

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-362-6

  • Online ISBN: 978-1-59745-000-3

  • eBook Packages: Springer Protocols

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