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

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

By: Antonin V. Tutter², Johannes C. Walter³

Abstract

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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.

Affiliation(s): (2) Novartis Institutes for Biomedical Research, Cambridge, MA
(3) Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA

Book Title: Xenopus Protocols: Cell Biology and Signal Transduction

Series: Methods in Molecular Biology | Volume: 322 | Pub. Date: Dec-13-2005 | Page Range: 121-137 | DOI: 10.1007/978-1-59745-000-3_9

Subject: Cell Biology

Key Words: Cell-free system - DNA replication - geminin - MCM2-7 - nucleoplasmic extract - ORC - origin unwinding - prereplication complex - Xenopus laevis

References

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