Extracts for Analysis of DNA Replication in a Nucleus-Free System

Frog egg extracts represent a powerful approach with which to dissect molecular mechanisms of vertebrate DNA replication and repair. In the classical approach, sperm chromatin is added to a crude egg lysate to form replication-competent nuclei. We subsequently described a procedure that bypasses the requirement for nuclear assembly in DNA replication. In this method, DNA is first added to a high-speed supernatant (HSS) of egg lysate, which mimics the G 1 phase of the cell cycle in that it supports replication licensing. Subsequent addition of a concentrated nucleoplasmic extract (NPE) leads to replication initiation followed by a single complete round of DNA replication. The advantage of the nucleus-free system is that it supports efficient replication of model DNA templates such as plasmids and lambda DNA that can be modified with specific features such as LacI arrays, DNA protein cross-links, or DNA interstrand cross-links. Here, we describe our current protocol for preparation of HSS and NPE. Methods for their use in DNA replication and repair are described elsewhere.