Replication of the Bacterial Chromosome: Location of New Initiation Sites After Irradiation

Abstract

New loci of replication along the bacterial chromosome are observed after irradiation of Escherichia coli. It was conjectured that, after X-irradiation, the new initiation site was random with respect to the fixed-origin, whereas, after ultraviolet light exposure, it was selective and appeared to be from the fixed-origin. Evidence presented here shows that, after X-irradiation of E. coli, the new initiation site(s) for the onset of deoxyribonucleic acid replication is induced at chromosomal regions not restricted to the fixed-origin. After ultraviolet light exposure, the new initiation site is preferentially from the fixed-origin. In these studies amino acid starvation was used to synchronize chromosome replication and to allow for differential radioisotopic labeling of the chromosomal origin and terminus. To facilitate interpretation, growing cells actively replicating their chromosome were compared with cells lacking growth points at the time of irradiation. The role of these new replication sites in the observed kinetics of deoxyribonucleic acid replication following X-ray or ultraviolet light exposure is discussed.An alteration in the expected sequence of chromosome replication in Escherichia coli occurs after exposure to ultraviolet light (UV; reference 13) or X-rays (7). The reorientation of the replication sequence is not an artifact of the methodology (14) or a consequence of repair synthesis (8), but results from a semiconservative synthesis starting at a region differing from that occupied by the growth point at the time of irradiation.The present studies were designed to determine whether the new initiation sites were at the fixedorigin of cycles of replication or elsewhere on the chromosome. Advantage was taken of the amino acid deprivation method which allows synchronization of replication starting from the vegetative origin (fixed-origin) of the bacterial chromosome (1,9,21,23). The sequence of replication of radioisotopically marked origin and terminus was then followed by density-transfer.The resulting data show that, in the immediate round of replication following X-irradiation, the new initiation sites are non-origin in location, whereas, after UV they are predominantly from the origin.MATERIALS AND METHODS Bacterial growth. The multiauxotrophic E. coli 15T-(555-7) requiring thymine, arginine, methionine, and tryptophan was used. The cells were grown with forced aeration at 37 C in a minimal salts medium (3) supplemented with 20 ,ug of arginine, 30 /g of methionine, 14 Mug of tryptophan, and 20 MAg of thymine per ml.Harvesting of cells. Cells were harvested and washed on membrane filters (Millipore Filter Corp., Bedford, Mass.) at ambient temperature to reduce trauma. Care was exercised to avoid drying the cells on the filter. The washing consisted of adding an equal volume of minimal salts solution followed by filtration. The sequence was repeated three times. The total time required for washing was less than 5 min.There was no observable deleterious effect on cell growth or macromolecular synt...

Temperature-Sensitive Initiation of Chromosome Replication in a Mutant of Escherichia coli

“…Irradiation with gamma-rays causes single-strand breaks (27), and Kohiyama proposes that these serve as new sites of initiation at 41 C. Densitytransfer experiments suggest that this initiation occurred at random positions on the chromosome, which is consistent with this proposal. Initiation at points other than the replication origin has also been observed in wild-type cells after X-ray irradiation (3).…”

Recombinational Repair of DNA Damage in Escherichia coli and Bacteriophage λ

“…If cells restarted DNA replication only when a "safe" level of DNA damage was attained as a result of repair, there would have been no effect of this drug-mediated inhibition of DNA synthesis on cell sur-vival. Finally, the idea that E. coli has a mechanism to inhibit replication of damaged DNA is incompatible with the observations that E. coli initiates extra rounds of DNA replication from the origin when its DNA is heavily damaged (46,300,501). All these phenomena seem to indicate that, in contrast to eukaryotes, E. coli lacks a mechanism to stop chromosomal replication when its DNA is damaged and instead relies on enhanced repair and damage tolerance in its attempt to faithfully replicate the damaged genome.…”

Instability of inhibited replication forks in E. coli

“…A mechanism for inducible stable DNA replication In addition to destabilizing replication forks, inhibiting the replication fork advance also initiates a new round of DNA replication at one of the two origins of the replication bubble. Such reinitiation is observed after thymine starvat i~n (~~) , UV irradiation (45) or incubation of dnaBts mutants at the non-permissive t e m p e r a t~r e s (~~1~~) .…”