In the previous funding period it was shown that: (a) Sparsely ionizing radiation induces at least two different forms of potentially lethal damage (PLD). The terms alpha-PLD and beta-PLD were proposed, respectively. (b) Similarities exist between alpha- PLD and split-dose recovery (SDR), suggesting a common molecular base for these cellular repair reactions. (c) Repair and fixation of alpha-PLD is involved in the variation in radiosensitivity observed through the cell cycle.
The aim of this proposal is to investigate the nature of the DNA lesions underlying PLD and to elucidate the mechanism(s) of fixation. The hypothesis is that abrupt changes in chromatin conformation naturally occurring during the postirradiation period affect otherwise repairable DNA lesions (probably dsb) causing damage fixation that results in lethal chromosome aberrations. These changes may occur with: (a) progression of cells through the cycle, especially before or after mitosis, and the changes that occur at the initiation of DNA synthesis; (b) induction of proliferation by subculture of plateau-phase cells; and (c) postirradiation incubation in anisotonic media. Local distortions in chromatin conformation, indirectly induced after postirradiation treatment with araA, may also cause PLD fixation. To test this hypothesis, experiments will be performed simultaneously with the same cell population at the DNA level using neutral filter elution, at the chromosome level using premature chromosome condensation (PCC), and at the survival level. Chinese hamster CHO-cells and a mutant subline deficient in repair of DNA double strand breaks will be used to study the importance of DNA dsb induction and repair in chromosome fragmentation and in PLD repair and fixation. Syrian hamster wildtype BHK 21 cells and a temperature sensitive mutant (tsBN2) with a defect in the mechanism controlling chromosome condensation, will be used to study the effect of chromatin condensation on chromosome fragmentation and PLD fixation in interphase cells. In tsBN2 cells chromatin condensation and PCC formation can be induced at any phase of the cycle by transfer to the non-permissive temperature of 40 degrees C. The effects of radiation will be studied during the transition from M to G1 phase, where dramatic changes occur in chromatin conformation, in CHO and XRS-5 cells and in various phases of the cell cycle in tsBN2 cells. The results obtained will be combined to: (a) elucidate the sequence of events from damage in the DNA to chromosome damage and, ultimately, to cell death; and (b) establish a relationship between damage fixation (chromosome, DNA and survival levels) and changes in chromatin conformation occurring postirradiation. Chromatin conformation will be monitored by microscopy using the PCC technique. By shifting the importance from repair to fixation processes, this hypothesis unifies a variety of cellular phenomena observed after irradiation, such as repair and fixation of alpha- PLD, SDR, and variation of radiosensitivity through the cell cycle.
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