Rudin N, Haber J E
Department of Biology, Brandeis University, Waltham, Massachusetts 02254.
Mol Cell Biol. 1988 Sep;8(9):3918-28. doi: 10.1128/mcb.8.9.3918-3928.1988.
Novel recombinational repair of a site-specific double-strand break (DSB) in a yeast chromosome was investigated. When the recognition site for the HO endonuclease enzyme is embedded in nonyeast sequences and placed between two regions of homology, expression of HO endonuclease stimulates recombination between the homologous flanking regions to yield a deletion, the apparent product of an intrachromosomal exchange between direct repeats. This deletion-repair event is very efficient, thus preventing essentially all the potential lethality due to the persistence of a DSB. Interestingly, unlike previous studies involving spontaneous recombination between chromosomal repeats, the recombination events stimulated by HO-induced DSBs are accompanied by loss of the sequences separating the homologous regions greater than 99.5% of the time. Repair is dependent on the RAD52 gene. The deletion-repair event provides an in vivo assay for the sensitivity of any particular recognition site to HO cleavage. By taking advantage of a galactose-inducible HO gene, it has been possible to follow the kinetics of this event at the DNA level and to search for intermediates in this reaction. Deletion-repair requires approximately 45 min and is inhibited when cycloheximide is added after HO endonuclease cleavage.
对酵母染色体中位点特异性双链断裂（DSB）的新型重组修复进行了研究。当HO核酸内切酶的识别位点嵌入非酵母序列并置于两个同源区域之间时，HO核酸内切酶的表达会刺激同源侧翼区域之间的重组，从而产生缺失，这显然是直接重复序列之间染色体内部交换的产物。这种缺失修复事件非常高效，因此基本上可以防止由于DSB持续存在而导致的所有潜在致死性。有趣的是，与之前涉及染色体重复序列之间自发重组的研究不同，HO诱导的DSB刺激的重组事件在超过99.5%的时间里伴随着同源区域之间间隔序列的丢失。修复依赖于RAD52基因。缺失修复事件为任何特定识别位点对HO切割的敏感性提供了一种体内检测方法。通过利用半乳糖诱导型HO基因，有可能在DNA水平上追踪这一事件的动力学，并寻找该反应中的中间体。缺失修复大约需要45分钟，并且在HO核酸内切酶切割后添加环己酰亚胺时会受到抑制。
