Initiation of DNA replication in Escherichia coli 15 T−: Chronological dissection of three physiological processes required for initiation

Abstract

The replicative origin of the Escherichia coli chromosome was labeled with [ 3H]thymine after amino acid starvation. Replication of this label was measured by further growth in [ 13C]glucose- 15NH 4Cl. Such replication served as a measure of the initiation of a new cycle of chromosome replication. A new replication cycle was initiated about 35 minutes after labeling the chromosome origin. Initiation was heterogeneous, occurring earlier in some cells than in others. Two synthetic processes were distinguished. One requires amino acids, and is inhibited by 150 μg chloramphenicol/ml. and by 0.25% phenethyl alcohol. It was not inhibited by 25 μg chloramphenicol/ml. This process was completed about 15 minutes before the actual re-initiation of replication. The other was inhibited by 25 μg of chloramphenicol/ml. and was completed 30 minutes before re-initiation occurred. A third requirement for initiation was suggested by the fact that initiation did not occur immediately after necessary protein synthesis was completed. Both of these processes limit stoichiometrically the initiation of replication since they must be repeated to allow initiation of a second cycle of replication. In the absence of thymine, cells acquired the ability to initiate a new replication cycle despite subsequent inhibition of protein synthesis. The time at which this potential was achieved was independent of DNA synthesis.