EVIDENCE FOR ACTIVITIES INHIBITING IN TRANS INITIATION OF DNA-REPLICATION IN EXTRACT PREPARED FROM IRRADIATED-CELLS

We have previously shown that replication in vitro of plasmids contain ing the Simian virus 40 (SV40) origin of replication is reduced when a n extract of irradiated cells is used (Wang et al., Radiat. Res. 142, 169-175, 1995). We proposed that the observed reduction in the overall replication activity is due to a reduction in the efficiency of initi ation events, and that it is caused by the induction or activation by ionizing radiation of a factor(s) that inhibits DNA replication in tra ns. Here, we extend these studies and provide evidence that the reduce d replication activity of an extract prepared from irradiated cells is not the result of a nonspecific inactivation of proteins or of an inc rease in the requirement for SV40 large tumor antigen (TAg), the only noncellular protein required for in vitro DNA replication. Mixing expe riments demonstrate the presence of a dominant inhibitory activity(ies ) in the extract of irradiated cells that efficiently stalls replicati on in reactions assembled using extract of nonirradiated cells. The in hibitory activity is a stable, nondialyzable molecule. Studies of kine tics suggest that the inhibitory activity(ies) affects the initiation steps of DNA replication and acts, at least partly, by modifying TAg, the key initiation protein of SV40 ori DNA replication. It is likely t hat the same inhibitory activity(ies) regulates cellular DNA replicati on by modifying the cellular homologues of TAg. Purification and chara cterization of this inhibitory activity(ies) will contribute to our un derstanding of the mechanism developed by the cell to regulate DNA rep lication after exposure to ionizing radiation and will define a checkp oint operating in S phase. Genetic evidence for a checkpoint in S phas e distinct from the checkpoints operating in G(1) and G(2) phase has b een reported in yeast. (C) 1996 by Radiation Research Society