36 DNA Replication in Xenopus

Abstract

Cell-free extracts of eggs of the South African clawed toad Xenopus laevis support complete chromosome replication under normal cell cycle control (Blow and Laskey 1986). Apart from a similar system derived from embryos of the fruit fly Drosophila melanogaster (Crevel and Cotterill 1991), it is currently the only eukaryotic cell-free system that supports efficient chromosome replication in vitro. Since the Xenopus cell-free system progresses through the complete cell cycle in vitro (Hutchison et al. 1987; Murray and Kirschner 1989), it offers a unique opportunity to study the way that DNA replication is coordinated with other cell-cycle events. This subject has recently been reviewed in detail (Blow 1996); the major conclusions are summarized here.

When Xenopus eggs are crushed by low-speed centrifugation, the resultant low-speed supernatants, which contain abundant particulate material (including nuclear envelope precursors), can support all the major activities of the early embryonic cell cycle. During each in vitro cell cycle there is an ordered sequence of events as follows:Activation of certain essential replication proteins and their assembly onto decondensing chromosomes

Assembly of chromosomal DNA into nuclei with an intact nuclear envelope

Import of proteins required for the initiation and progression of replication

Termination of replication forks and inactivation of certain replication proteins to prevent re-replication of DNA in the current cell cycle.

THE ROLE OF NUCLEAR ASSEMBLY IN DNA REPLICATION
The Xenopus system can replicate a wide range of different DNA templates, including demembranated Xenopus sperm nuclei (sperm chromatin: the natural substrate for...