Radiation down-regulates replication origin activity throughout the S phase in mammalian cells

An asynchronous culture of mammalian cells responds acutely to ionizing rad iation by inhibiting the overall rate of DNA replication by similar to 50% for a period of several hours, presumably to al low ti me to repair DNA dam age. At low and moderate doses, this S phase damage-sensing (SDS) pathway a ppears to function primarily at the level of individual origins of replicat ion, with only a modest inhibition of chain elongation parse We have shown previously that the majority of the inhibition observed in an asynchronous culture can be accounted for by late G(1) cells that were within 2-3 h of e ntering the S period at the time of irradiation and which then fail to do s o. A much smaller effect was observed on the overall rate of replication in cells that had already entered the S phase. This raised the question wheth er origins of replication that are activated within S phase per se are inhi bited in response to ionizing radiation. Here we have used a two-dimensiona l gel replicon mapping strategy to show that cells with an intact SDS pathw ay completely down-regulate initiation in both early- and late-firing rDNA origins in human cells. We also show that initiation in mid- or late-firing rDNA origins is not inhibited in cells from patients with ataxia telangiec tasia, confirming the suggestion that these individuals lack the SDS pathwa y.