Initiation of DNA replication in Bacillus subtilis. IV. The effect of an intercalating dye, ethidium bromide

Summary The effects of an intercalating dye, ethidium bromide (EtBr), on the initiation of chromosome replication in Bacillus subtilis were studied. Spores of a thymine requiring mutant acquired the ability to initiate one round of replication in the absence of RNA and protein synthesis (initiation potential) during germination in a thymine starved medium. When EtBr was added after the initiation potential was fully established, initiation of replication was completely inhibited. This inhibition was reversible, and initiation was resumed when the drug was removed. The recovery of initiation occurred in the absence of protein synthesis but did require RNA synthesis and an active dna gene product.During germination both a DNA-protein complex and a DNA-membrane complex were formed at the replication origin in parallel with the establishment of initiation potential. EtBr destroyed both of these complexes at the concentration which inhibited initiation.The first round of replication of a plasmid DNA, pSL103, during spore germination was also prevented by EtBr. However a higher concentration was required to inhibit plasmid replication. It was found that the plasmid formed two complexes identical to the S- and M-complex of the chromosome origin. Compared to the chromosome complexes the plasmid complexes were less sensitive to EtBr. The loss of sensitivity was equivalent to that for the initiation of the plasmid compared to the chromosome. These results indicate that the target of EtBr is the DNA in the S- and M-complexes whose conformation is essential for the initiation of chromosome and plasmid replication.III of this series is Murakami et al. 1976     

Discontinuity in DNA replication during expression of accumulated initiation potential in dnaA mutants of Escherichia coli.

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 degrees 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 min, 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 round 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 microgram/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 first 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.     

DNA replication initiation by 6-DMAP treatment in maturing oocytes and dividing embryos from marine invertebrates

6-dimethylaminopurine (6-DMAP), a potent protein kinase inhibitor, drives most cells into an interphasic stage. Experiments were undertaken with oocytes from three marine invertebrate species, i.e., Mytilus edulis, Spisula solidissima, and Strongylocentrotus droebachiensis, wherein oocytes were arrested at different phases of meiosis. 6-DMAP induced a continuous DNA synthesis in meiotic cells, whereas it allowed a single round of DNA replication in treated mitotic cells, regardless of species considered. The effects of 6-DMAP were accompanied in all cases by rephosphorylation on tyrosine of the p34^cdc2 homolog, the M-phase promoting factor (MPF) catalytic subunit. The fact that 6-DMAP overcomes the inhibitory control of replication during meiosis suggests that this process depends upon protein phosphorylation, while DNA synthesis regulation in mitotic cells relies on 6-DMAP-insensitive events. © 1996 Wiley-Liss, Inc.     

Role of the cgtA gene function in DNA replication of extrachromosomal elements in Escherichia coli

The cgtA gene codes for a common GTP-binding protein whose homologues were found in all prokaryotic and eukaryotic organisms investigated so far. Although cgtA is an essential gene in most bacterial species, its precise functions in the regulation of cellular processes are largely unknown. In Escherichia coli, dysfunction or overexpression of the cgtA gene causes problems in various chromosomal functions, like synchronization of DNA replication initiation and partitioning of daughter chromosomes after a replication round. It is not know how the cgtA gene product regulates these processes. Here we investigated effects of cgtA dysfunction on replication of plasmid and phage replicons. We found that replication of some plasmids (e.g., ColE1-like) is not affected in the cgtA mutant. On the other hand, dysfunction of the cgtA gene caused a strong inhibition of lambda plasmid DNA replication. Bacteriophage lambda development was severely impaired in the cgtA mutant. Replication of other plasmid replicons (derivatives of F, R1, R6K, and RK2) was influenced by the cgtA mutation moderately. It seems that DNA synthesis per se is not affected by CgtA, and that this protein might control replication initiation indirectly, by regulation of function(s) or production of one or more replication factors. In fact, we found that level of the host-encoded replication protein DnaA is significantly decreased in the cgtA mutant. This indicates that CgtA is involved in the regulation of dnaA gene expression.