REGULATION OF DNA-REPLICATION IN IRRADIATED-CELLS BY TRANS-ACTING FACTORS

We compared DNA replication activity in cytoplasmic extracts prepared from irradiated and nonirradiated HeLa cells using a simian virus 40 ( SV40)-based in vitro replication assay. The assay measures semi-conser vative DNA replication in a plasmid carrying the SV40 origin of replic ation and requires SV40 T antigen as the sole noncellular protein. The plasmid DNA used in the replication reaction is never exposed to radi ation. We find that replication of plasmid DNA is significantly reduce d when cytoplasmic extracts from irradiated cells are used, Since plas mid replication proceeds to completion in extracts from irradiated cel ls, the observed reduction in the over-all replication activity is pro bably due to a reduction in the efficiency of initiation events. The d egree of inhibition of DNA replication after exposure to 10, 30 and 50 Gy X rays as measured ill vitro using this assay is similar to that m easured in intact cells immediately before processing for extract prep aration. These observations are compatible with the induction or activ ation by ionizing radiation of a factor(s) that inhibits in trans DNA replication. The results contribute to our understanding of the mechan ism(s) developed by the cells to regulate DNA replication when exposed to clastogenic agents. Such processes may be of significance in the r estoration of DNA integrity, and may define yet another checkpoint ope rating during S at the level of clusters of replicons. (C) 1995 by Rad iation Research Society