Repair and recombination of X-irradiated plasmids in Xenopus laevis oocytes
Repair and recombination of X-irradiated plasmids in Xenopus laevis oocytes
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DOI：
10.1128/mcb.10.11.5849-5856.1990
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发表时间：
1990-11
影响因子：
5.3
通讯作者：
S. Sweigert;D. Carroll
中科院分区：
文献类型：
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作者：
S. Sweigert;D. Carroll
将质粒DNA底物经X射线照射并注射到非洲爪蟾卵母细胞的细胞核中。孵育20小时后，从卵母细胞中回收DNA，并同时分析修复和通过电泳和细菌转化进行的分子间同源重组。卵母细胞介导的DNA链断裂的修复观察与这两种方法。使用修复缺陷的突变体大肠杆菌菌株及其修复精通的父母作为宿主的转化试验，我们还表明，卵母细胞修复氧化型DNA碱基损伤的X射线。X射线照射的环状DNA刺激其潜在的重组与同源线性合作伙伴。通过Southern印迹杂交直接检测纯化产物，并且作为表达两种抗生素抗性标记的细菌纯化克隆，所述两种抗生素抗性标记最初分别携带在两种底物上。复合的增加依赖于X射线剂量。有一些建议，病变以外的双链断裂有助于刺激卵母细胞介导的同源重组。总之，卵母细胞具有相当大的修复X射线损伤的能力，并且某些X射线损伤刺激这些细胞中的同源重组。
Plasmid DNA substrates were X-irradiated and injected into the nuclei of Xenopus laevis oocytes. After incubation for 20 h, DNA was recovered from the oocytes and analyzed simultaneously for repair and for intermolecular homologous recombination by electrophoresis and bacterial transformation. Oocyte-mediated repair of DNA strand breaks was observed with both methods. Using a repair-deficient mutant Escherichia coli strain and its repair-proficient parent as hosts for the transformation assay, we also demonstrated that oocytes repaired oxidative-type DNA base damage induced by X-rays. X-irradiation of a circular DNA stimulated its potential to recombine with a homologous linear partner. Recombination products were detected directly by Southern blot hybridization and as bacterial transformant clones expressing two antibiotic resistance markers originally carried separately on the two substrates. The increase in recombination was dependent on X-ray dose. There is some suggestion that lesions other than double-strand breaks contribute to the stimulation of oocyte-mediated homologous recombination. In summary, oocytes have considerable capacity to repair X-ray-induced damage, and some X-ray lesions stimulate homologous recombination in these cells.