Down-regulation of DNA replication in extracts of camptothecin-treated cells: activation of an S-phase checkpoint?

Extracts prepared from camptothecin (CPT)-treated cells have a reduced ability to support SV40 DNA replication in vitro. This reduction derives mainly from a reduction in the frequency of initiation events because DNA chain elongation remains practically unchanged. Mixing of extract from nontreated cells with small amounts of extract of CPT-treated cells indicates that the reduction in DNA replication is due to the synthesis/activation of a dominant inhibitor. The observed reduction in DNA replication activity cannot be attributed to inactivation of Topo I, the molecular target of camptothecin, because levels and activity of this protein remain unchanged in extracts of CPT-treated cells and addition of purified Topo I does not restore replication activity. Although replication protein A (RP-A) is phosphorylated in CPT-treated cells, reduced replication may not be caused by RP-A inactivation, because neither loss of phosphorylation nor the addition of recombinant RP-A restore replication activity. We interpret these observations as biochemical evidence for the activation of a checkpoint in S phase and discuss the ramifications of this activation on the mechanism of CPT-induced cytotoxicity.