RADIATION EFFECTS ON DNA-SYNTHESIS IN A DEFINED CHROMOSOMAL REPLICON

It has recently been shown that the tumor suppressor p53 mediates a si gnal transduction pathway that responds to DNA damage by arresting cel ls in the late G(1) period of the cell cycle. However, the operation o f this pathway alone cannot explain the 50% reduction in the rate of D NA synthesis that occurs within 30 min of irradiation of an asynchrono us cell population. We are using the amplified dihydrofolate reductase (DHFR) domain in the methotrexate-resistant CHO cell line, CHOC 400, as a model replicon in,which to study this acute radiation effect. We first show that the CHOC 400 cell line retains the classical acute-pha se response but does not display the late G(1) arrest that characteriz es the p53-mediated checkpoint. Using a two-dimensional gel replicon-m apping method, we then show that when asynchronous cultures are irradi ated with 900 cGy, initiation in the DHFR locus is completely inhibite d within 30 min and does not resume for 3 to 4 h. Since initiation in this locus occurs throughout the first 2 h of the S period, this resul t implies the existence of a p53-independent S-phase damage-sensing pa thway that functions at the level of individual origins. Results obtai ned with the replication inhibitor mimosine define a position near the G(1)/S boundary beyond which cells are unable to prevent initiation a t early-firing origins in response to irradiation. This is the first d irect demonstration at a defined chromosomal origin that radiation qua ntitatively down-regulates initiation.