Potential for initiation of chromosome replication present in temperature-sensitive, initiation-defective dnaA5 mutants of Escherichia coli B/r incubated at nonpermissive temperature was expressed by shifting to a more permissive temperature (25°C). Upon expression of initiation potential, the rate of [3H]thymidine incorporation varied in a bimodal fashion, i.e., there was an initial burst of incorporation, which lasted 10 to 20 minm then a sudden decrease in incorporation, and finally a second rapid increase in incorporation. Analyses of this incorporation pattern indicated that a round of replication initiated upon expression of initiation potential, but DNA polymerization stopped after replication of 5 to 10% of the chromosome. This t-ound of replication appeared to resume about 30 min later coincident with initiation of a second round of replication. The second initiation was unusually sensitive to low concentrations of novobiocin (ca. 1 Fg/ml) when this inhibitor was added in the presence of chloramphenicol. In the absence of chloramphenicol, novobiocin at this concentration had no detectable effect on DNA replication. It is suggested that cis-acting inhibition, attributable to an attempted second initiation immediately after the first, caused the ftirst round to stall until both it and the second round could resume simultaneously. This DNA replication inhibition, probably caused by overinitiation, could be a consequence of restraints on replication in the vicinity of oriC, possibly topological in nature, which limit the minimum interinitiation interval in E. coli. round of replication, the first initiation event is followed by additional initiation events, with the time interval between the first and second events being about 30 min at 25 to 30°C (6, 7, 13). The initiation events require RNA polymerase activity for expression (7,13,15,25,36) and, apparently, a critical chromosomal superhelicity which could be needed for a transcriptional step or for other steps in initiation or both (9)(10)(11)27). It has recently been suggested that the time interval between initiations is a function, at least in part, of the time required for the newly replicated oriC to be methylated (24). Minichromosomes, i.e., plasmids which initiate replication from a resident copy of ariC, behave very similarly to chromosomes with respect to accumulation and expression of initiation potential in dnaA mutants (18,20).In this paper we examine the detailed kinetics of replication of chromosomes and minichromosomes upon expression of initiation potential. A bimodal pattern of DNA replication is reported. The possible causes of this unusual pattern are examined and discussed.
MATERIALS AND METHODSBacteria and growth conditions. The organisms used were E. coli B/r F62 (dnaA5 thyA his) and E. coli B/r F621 (dnaAS his recA). Strain B/r F621 contained the minichromosome pAL2, which consists of a 1.3-megadalton DNA fragment containing the chromosomal origin of replication (oriC) and a 5.7-megadalton fragment containing a kanamycin resistance ...