Sedimentation of DNA of Dictyostelium discoideum lysed on alkaline sucrose gradients: Role of single-strand breaks in gamma ray lethality of sensitive and resistant strains

Abstract

The nuclear and mitochondrial DNA of the amoebae of the cellular slime mold Dictyostelium discoideum have been labeled with [ methyl- 3H]thymidine by allowing them to grow on Escherichia coli 15T − containing this label in its DNA. Neutral CsCl gradients were used to identify the labeled molecules. Alkaline sucrose sedimentation profiles of cells lysed directly on the gradients revealed two high molecular weight species, one of about 90 S (single-strand mol wt = 1.4 × 10 8) identified by alkaline CsCl rebanding as nuclear DNA, and another of 43 S (single-strand mol wt = 2.3 × 10 7), identified as mitochondrial DNA. These alkaline sucrose gradients were used to study the production of single-strand breaks and their rejoining in DNA of a gamma ray-resistant strain (NC-4; 10% survival dose for cell proliferation, D 10 = 300 krad) and in two radiation-sensitive daughter mutants (γs-18, D 10 = 75 krad; γs-13, D 10 = 4 krad). With 60Co gamma rays, breaks were produced in nuclear and mitochondrial DNA at an efficiency of one break per 33 eV in all three strains. At doses up to about 100 krad, these single-strand breaks were closed equally well during post-irradiation incubation of NC-4, γs-18 and γs-13, even though their survivals were widely different, indicating no apparent correlation between parental strand rejoining and survival in the sensitive strains. At higher doses, post-irradiation treatment with 1 mg caffeine/ml sensitized NC-4 and retarded strand-rejoining, suggesting that lethality in this resistant strain may be related to strand breaks. It is concluded that single-strand rejoining is a necessary, but not sufficient, condition for radiation survival in this organism. The nature of the apparently unrepaired lesions leading to lethality in the sensitive strains is not known.