Intermediates in extrachromosomal homologous recombination in Xenopus laevis oocytes: characterization by electron microscopy.
Intermediates in extrachromosomal homologous recombination in Xenopus laevis oocytes: characterization by electron microscopy.
复制标题
非洲爪蟾卵母细胞染色体外同源重组的中间体：电子显微镜表征。
DOI：
10.1002/j.1460-2075.1993.tb05628.x
复制
发表时间：
1993
期刊：
影响因子：
--
通讯作者：
Carroll,D
中科院分区：
文献类型：
--
作者：
Pont-Kingdon,G;Dawson,RJ;Carroll,D
已经提出了几种分子机制来解释非保守同源重组。当注入适当的DNA底物时，这种类型的重组在非洲爪蟾卵母细胞中特别有效。为了区分可能的模型，我们在电子显微镜下通过直接观察研究了卵母细胞的重组中间体。部分重组的DNA在卵母细胞中孵育后与补骨脂素衍生物交联，以限制在恢复过程中可能发生的分支迁移，并改变最初存在的结构。分支结构，我们解释为中间产物，代表了大约10%的DNA恢复，并很容易分析。我们没有观察到任何由入侵机制预测的具有内部环的结构。大多数中间体在产物大小的分子上有一个或两个单链分支，正如在切除-退火机制中预测的不完全连接。详细的长度测量证实了该模型的预期。当恢复的DNA没有交联，或者在体外制备退火连接时，我们看到分支结构，表明发生了广泛的分支迁移。与交联样品的比较证实了交联在保存卵母细胞中形成的结构方面的有效性。我们的结果有力地支持了卵母细胞重组的切除-退火机制。
Several molecular mechanisms have been proposed to account for nonconservative homologous recombination. This type of recombination is particularly efficient in Xenopus oocytes when appropriate DNA substrates are injected. To distinguish between possible models, we have investigated recombination intermediates from oocytes by direct observation in the electron microscope. Partially recombined DNA was crosslinked with a psoralen derivative after incubation in oocytes to limit the branch migration that might occur during recovery procedures and alter the structures that were initially present. Branched structures, which we interpret as intermediates, represented approximately 10% of the DNA recovered and were readily analyzed. We did not observe any structures with internal loops predicted by invasion mechanisms. The majority of intermediates had one or two single‐stranded branches on product‐sized molecules, as predicted for incomplete junctions in the resection‐annealing mechanism. Detailed length measurements confirmed the expectations of that model. When recovered DNA was not crosslinked, or when annealed junctions were prepared in vitro, we saw branched structures that indicated the occurrence of extensive branch migration. Comparison with the crosslinked sample confirmed the effectiveness of the crosslinking in preserving structures created in the oocytes. Our results strongly support a resection‐annealing mechanism of recombination in oocytes.