Ionizing radiation blocks DNA chain elongation in normal diploid fibroblasts but not in fibroblasts from patients with ataxia-telangiectasia, even though there are no differences in the damage induced between the two cell types. This difference suggests that radiation-induced lesions in DNA are not themselves blocks to chain elongation in ataxia cells and raises the possibility that in normal cells a mediator exists between DNA damage and chain termination.

Normal human cells and cells from patients with ataxia-telangiectasia (A-T) were exposed to culture medium made hypertonic by raising the NaCl concentration. The rate of DNA synthesis in both types of cells was depressed as a function of increasing hypertonicity. When cells of both types were exposed to X radiation and incubated in hypertonic medium, DNA synthesis appeared to be more radioresistant than in cells incubated in normal medium. Velocity sedimentation analysis showed that this was due to a hypertonicity-induced inhibition of replicon initiation, which is the same process affected by X radiation, indicating that the two treatments were not additive. After a 5-hr incubation in hypertonic medium, there was a new steady state of replicon initiation and elongation similar to that existing in cells grown in normal medium, except that fewer replicons were participating. At this time DNA synthesis in each type of cell had a characteristic response to radiation, i.e., radiosensitive in normal cells and radioresistant in A-T cells. These results suggest that radioresistant DNA synthesis in A-T cells is not due to increased condensation of chromatin.

The changes in sedimentation distance of nucleoids in neutral sucrose gradients in the presence of various concentrations of the intercalating dye ethidium bromide are believed to reflect changes in the amount of DNA supercoiling within the nucleoid. When HeLa cells were heated to 45°C for 30 min before cell lysis, sedimentation of their nucleoids was markedly affected; at all concentrations of ethidium bromide in the gradient, nucleoids from heated cells sedimented farther than those from unheated cells. After cellular protein was labeled with [ 3 H]leucine for 15 to 17 hr, approximately 4% of the total 3 H radioactivity cosedimented with nucleoids from unheated cells. This percentage increased with heating time (10-70 min) and temperature (44-46°C); e.g., after 20 min at 45°C the fraction of 3 H cosedimenting with nucleoids was 7%. The heat-induced change in nucleoid sedimentation distance was a linear function of the percentage of 3 H cosedimenting with nucleoids. These results show that hyperthermia causes an increase in the protein content of nucleoids. The additional protein affects sedimentation by increasing the mass of the nucleoids and by decreasing the amount of DNA available for supercoiling and thereby the efficiency of DNA rewinding.

About 20% of the single-strand breaks produced by X-rays in V79 Chinese hamster cells under extremely hypoxic conditions remain unrepaired after 1 hr at 37°C. The corresponding figure is only 10% for cells irradiated under moderately hypoxic (0.2 μM) or aerobic conditions (200 μM). The initial number of breaks produced per unit dose is identical for extremely hypoxic and moderately hypoxic cells and is about four times greater for aerobic cells. Because split-dose recovery proceeds under aerobic and moderately hypoxic conditions and is inhibited only under extremely hypoxic conditions, the split-dose recovery phenomenon may be related to the unrepaired component of single-strand breaks.

Repair replication was measured after uv-irradiation of WI-38 cells at different passage numbers. A reduced level of DNA synthesis was found only at the last passage before cessation of cell division. This reduction was not evident two passages before this and we conclude that excision repair deficiency is not the basis for in vitro aging of diploid human cells.