Initiation of DNA replication cycles in Escherichia coli following DNA synthesis inhibition

Abstract

The pattern of initiation of new growing points in the bacterial chromosome has been studied in a temperature-sensitive DNA replication-deficient mutant of Escherichia coli, strain TAU-bar t3. In particular, the effect of a period at the restrictive temperature upon this pattern has been examined. Selected regions of the chromosome were marked by radioisotopic labeling in cultures synchronized by amino acid starvation. The subsequent pattern of DNA replication was determined by equilibrium sedimentation of isolated DNA in a CsCl density gradient after a period of DNA synthesis in the presence of 5-bromouracil. The relative amount of label in the hybrid 5-bromouracil DNA band indicated the replication of the marked regions. New growing points were preferentially initiated at the chromosomal origin, as defined by amino acid starvation, following a period of DNA synthesis inhibition. A period of inhibition obtained by placing the mutant at the restrictive temperature had the identical effect to that of a period of thymine starvation upon the subsequent premature initiation of new replication cycles. Our results support the hypothesis that the initiation of new cycles is independent of the presence or locations of existing points of replication and that it is dependent entirely upon cytoplasmic factors, such as protein synthesis and possibly membrane synthesis.