Fonck K, Barthel R, Bryant P E
Mutat Res. 1984 Sep-Oct;132(3-4):113-8. doi: 10.1016/0167-8817(84)90005-1.
It is known that mammalian cells repair X-ray-induced double-strand breaks (DSB). The mechanism of this repair is, however, as yet unknown but it is thought that the repair may involve recombination between homologous DNA strands. We have investigated the presence and kinetics of occurrence of recombinational molecular intermediates or 'heavy-heavy' (HH) hybrids in DNA of X-irradiated mouse Ehrlich ascites tumour cells unifiliarly substituted with bromodeoxyuridine. Purified DNA from density-labelled cells was analysed using isopyknic CsCl density centrifugation. After rebanding of the high-density material, the relative amount of HH-hybrid material was determined. When the cells were incubated after X-ray exposure, hybrids accumulated with an apparently biphasic kinetic; first a rapid accumulation directly after irradiation followed by a second, more slowly appearing peak. When DSB repair was inhibited by the nucleoside analogue 9-beta-D-arabinofuranosyladenine (ara-A), a powerful DNA polymerase inhibitor, the kinetics of the HH-hybrid formation were similar to those during the incubation after X-irradiation without ara-A. We interpret this as indicating that the first step in repair of DSB i.e. hybrid formation, occurs in the absence of DNA synthesis as predicted by the Resnick model of DSB repair. In an experiment in which the ara-A was washed away from the irradiated cells after 8-h treatment with the drug and replaced by fresh growth medium, so allowing DSB to repair, a similar HH-hybrid kinetic response was found to that occurring directly after irradiation in the absence of ara-A. The reasons for this are not yet clear. In this case, however, the response of the ara-A-treated cells after X-irradiation was much stronger than that in the untreated cells where only approximately 20% of the DSB remained. These kinetic results which show a temporary appearance of HH hybrids, indicate that a net exchange of genetic material does not take place; they therefore lend support to the postulated recombinational model of Resnick in which a temporary exchange between homologous DNA strands takes place during DSB repair.
已知哺乳动物细胞可修复X射线诱导的双链断裂（DSB）。然而，这种修复机制尚不清楚，但据认为该修复可能涉及同源DNA链之间的重组。我们研究了单链用溴脱氧尿苷取代的X射线照射的小鼠艾氏腹水瘤细胞DNA中重组分子中间体或“重-重”（HH）杂种的存在及出现动力学。使用等密度CsCl密度离心法分析来自密度标记细胞的纯化DNA。对高密度物质进行再带谱分析后，测定HH杂种物质的相对量。当细胞在X射线照射后进行孵育时，杂种以明显的双相动力学积累；首先在照射后立即快速积累，随后出现第二个较慢出现的峰值。当用核苷类似物9-β-D-阿拉伯呋喃糖基腺嘌呤（ara-A，一种强大的DNA聚合酶抑制剂）抑制DSB修复时，HH杂种形成的动力学与无ara-A的X射线照射后孵育期间的动力学相似。我们将此解释为表明DSB修复的第一步即杂种形成，是在DNA合成不存在的情况下发生的，这与DSB修复的雷斯尼克模型预测一致。在一项实验中，在用该药物处理8小时后将ara-A从照射的细胞中洗脱，并用新鲜生长培养基替代，从而使DSB得以修复，发现HH杂种动力学响应与无ara-A照射后直接出现的情况相似。其原因尚不清楚。然而，在这种情况下，ara-A处理的细胞在X射线照射后的反应比未处理的细胞强烈得多，未处理细胞中仅约20%的DSB残留。这些显示HH杂种暂时出现的动力学结果表明没有发生遗传物质的净交换；因此它们支持了雷斯尼克假设的重组模型，即在DSB修复过程中同源DNA链之间发生暂时交换。
