Double Holliday junctions are intermediates of DNA break repair.
Double Holliday junctions are intermediates of DNA break repair.
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DNA双链断裂（DSBs）的同源重组修复对细胞增殖和肿瘤抑制至关重要。然而，尽管它很重要，有丝分裂dsb修复的分子中间体仍然不明确。双霍利迪结（dHJ），假定是中心中间体超过25年，只在减数分裂重组中被确定。此外，已经积累的证据表明，dhj不依赖于其他机制，这提高了dhj在有丝分裂循环细胞的dsb修复过程中不形成的可能性。在这里，我们使用一种芽殖酵母试验系统来识别DSB修复的中间体，该系统旨在模拟DSB的生理修复。该系统利用二倍体细胞，提供了在姐妹染色单体之间或同系物之间进行等位基因重组的可能性，以及在同一位点与减数分裂重组的直接比较。在有丝分裂循环细胞中，我们检测到同源间关节分子（JM）中间体，其大小和链组成与减数分裂中观察到的典型dHJ结构相同。然而，与减数分裂相反，姐妹染色单体之间的JMs比同源染色单体之间的JMs更容易形成。此外，JMs似乎代表了有丝分裂细胞中DSB修复的一个次要途径，在减数分裂重组中检测到的水平（每个DSB）比减数分裂重组低约10倍。因此，尽管dhj在有丝分裂细胞和减数分裂细胞中被鉴定为dsb促进重组的中间产物，但它们的形成明显受到两种细胞程序的特定指令的调节。
Repair of DNA double-strand-breaks (DSBs) by homologous recombination is crucial for cell proliferation and tumor suppression. However, despite its importance, the molecular intermediates of mitotic DSB-repair remain undefined. The double Holliday Junction (dHJ), presupposed to be the central intermediate for more than 25 years, has only been identified during meiotic recombination. Moreover, evidence has accumulated for alternative, dHJ-independent mechanisms, raising the possibility that dHJs are not formed during DSB-repair in mitotically cycling cells. Here we identify intermediates of DSB-repair using a budding yeast assay system designed to mimic physiological DSB repair. This system utilizes diploid cells and provides the possibility for allelic recombination either between sister-chromatids or between homologs, as well as direct comparison with meiotic recombination at the same locus. In mitotically cycling cells, we detect inter-homolog Joint Molecule (JM) intermediates whose size and strand-composition are identical to the canonical dHJ structures observed in meiosis. However, in contrast to meiosis, JMs between sister chromatids form in preference to those between homologs. Moreover, JMs appear to represent a minor pathway of DSB repair in mitotic cells, being detected at ~10-fold lower levels (per DSB) than during meiotic recombination. Thus, although dHJs are identified as intermediates of DSB-promoted recombination in both mitotic and meiotic cells, their formation is distinctly regulated according to the specific dictates of the two cellular programs.
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