Analysis of inhibition of DNA replication in irradiated cells using the SV40-based in vitro assay of DNA replication

The mechanisms of inhibition of DNA replication after DNA damage through the activation of the S-phase checkpoint have been the focus of several investigations over the last 40 yr. Recent studies have identified several components of this checkpoint response and there is strong interest in its biochemical characterization. Helpful for the delineation of the mechanism of the S-phase checkpoint is the observation that factors inhibiting DNA replication in vivo can be found in active form in extracts prepared from irradiated cells, when these are tested using the simian virus 40 (SV40) assay for in vitro DNA replication. In this assay, replication of plasmids carrying the minimal origin of SV40 DNA replication is achieved in vitro using cytoplasmic cell extracts and SV40 large tumor antigen (TAg) as the only noncellular protein. Here, we describe protocols developed to measure in vitro DNA replication with the purpose of analyzing its regulation after exposure to DNA damage. The procedures include the preparation of components of the in vitro DNA replication reaction including cytoplasmic extracts from cells that have sustained DNA damage. The assay is powerful but is limited by the fact that initiation steps carried out by the TAg in vitro may have different cellular determinants.