CELLULAR REPAIR PLD AND SLD--COMMON MOLECULAR BASE

在上一个供资期间，表明：(A)稀少 电离辐射会导致至少两种不同形式的 潜在致命伤害(PLD)。术语α-PLD和β-PLD 分别提出了两种不同的分类方法。(B)α-之间存在相似之处 PLD和分裂剂量恢复(SDR)，提示一种共同的分子 这些细胞修复反应的基础。(C)修理和固定 α-PLD参与辐射敏感性的变化 通过细胞周期观察。 这项提议的目的是调查DNA的性质 PLD的基础病变及阐明其发病机制(S) 定格。假设是染色质的突然变化 辐射后自然形成的构象 影响本来可以修复的DNA损伤(可能是DSB)，导致 损伤固定会导致致命的染色体异常。 这些变化可能发生在：(A)细胞通过 周期，尤其是在有丝分裂之前或之后，以及 在DNA合成开始时发生；(B)诱导 平台期细胞继代培养增殖；及(C) 辐照后在非等渗介质中的孵化。局部扭曲 在染色质构象中，辐射后间接诱导 使用ARAA治疗，也可能导致PLD固定。为了测试这一点 假设，实验将与 在DNA水平上使用中性过滤洗脱的相同细胞群， 在染色体水平上使用过早的染色体凝聚 (PCC)，并在生存层面上。中国仓鼠的CHO细胞和 DNA双链断裂修复缺陷的突变亚系 将被用来研究DNA DSB诱导和 染色体断裂修复和PLD修复和固定。 叙利亚仓鼠野生型BHK-21细胞及温度敏感型 控制机制有缺陷的突变体(TsBn2) 染色体凝集，将被用来研究其影响 染色质凝聚对染色体碎裂和PLD固定的影响 在间期细胞中。TsBN_2细胞中染色质凝聚和PCC 可以在周期的任何阶段通过转移到 40摄氏度的不允许温度。 辐射将在从M相到G1相的转变过程中进行研究， 染色质构象发生显著变化的地方，Cho和 XRS-5细胞和tsBN2细胞处于细胞周期的不同时相。 所得结果将结合起来：(A)阐明 从DNA损伤到染色体损伤的一系列事件， 最终，导致细胞死亡；和(B)建立 损伤固定(染色体、DNA和存活水平)和变化 在染色质构象中，发生在辐射后。染色质 构象将通过使用PCC的显微镜进行监测 技术。通过将重点从修复转移到固定 过程，这一假说统一了多种细胞现象 照射后观察，如修复和固定α- PLD、SDR和放射敏感性在细胞周期中的变化。

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.