The effects of inhibition of protein synthesis by the antibiotics cycloheximide and puromycin on the initiation of DNA replication in mouse L cells were studied. Cellular DNA was pulse labeled with [3H]thymidine of high, then of low specific activity and prepared for fiber autoradiography. Autoradiograms containing multiple (up to four) replication units were analyzed. In control cells, the proportion of replication units that initiated during a 10-min, high specific activity pulse was approximately equal to the proportion initiating immediately before the pulse. The addition of cycloheximide or puromycin at the start of the pulse inhibited the frequency of initiation in that there was a decrease by up to one-third of units initiating during the pulse relative to controls. Replication direction was also altered. Addition of the antibiotics 2 h before the pulse reduced the proportion of bidirectional units observed from 0.98 to 0.70. Antibiotic treatment for 2 h also decreased initiation synchrony in that the proportion of multiunit autoradiograms on which neighboring units showed similar replication patterns (indicating temporally coordinated initiation) was reduced by one-half. These observations indicate that inhibition of protein synthesis alters the normal pattern of DNA initiation.

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