Neutral sucrose gradient sedimentation of very large DNA from Bacillus subtilis: II. Double-strand breaks formed by gamma ray irradiation of the cells

Abstract

A procedure has been developed whereby essentially all the DNA from Bacillus subtilis cells can be reproducibly extracted in a form which sediments 2.3 times faster than bacteriophage T2 DNA in a neutral sucrose gradient spun at 20,000 revs/ min. When the cells are irradiated with low (3 to 34 kilorads) gamma ray doses, some DNA moves in a slower peak, which from the previous paper (Levin & Hutchinson, 1973) appears to be linear DNA. Some of the DNA also sediments ahead of the unirradiated DNA. On incubation of the cells at 37°C under conditions such that single-strand DNA breaks are repaired, the fast-sedimenting component is partially restored, with the DNA sedimenting ahead of it usually disappearing, and the quantity in the slower component decreasing. With 80 minutes incubation the fraction of DNA after various radiation doses in the fast-sedimenting component is the same as the fraction of cells able to form colonies, suggesting that the destruction of the component is responsible for the effect of gamma rays on the ability of a cell to replicate. Single-strand breaks introduced into the DNA within the cells do not affect the fast-sedimenting component, so radiation-induced single-strand breaks are not responsible for the effect of gamma rays on replication. The double-strand break rate for DNA in the cells is 0.010 breaks per mass the size of T2 DNA per kilorad. The fast-sedimenting component in irradiated cells which have not been incubated disappears at a rate equal to one radiationinduced double-strand break formed per genome. Since the fast-sedimenting component in solution is also destroyed by one double-strand break per genome (Levin & Hutchinson, 1973), it is suggested that this component is the genome in the form of a circle. The correspondence between DNA in the fast-sedimenting form after incubation and the ability of cells to form colonies then indicates that a genome can replicate only if all double-strand breaks are repaired.