Genetic and physical analysis of double-strand break repair and recombination in Saccharomyces cerevisiae.
Genetic and physical analysis of double-strand break repair and recombination in Saccharomyces cerevisiae.
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发表时间：
1989-07
期刊：
影响因子：
3.3
通讯作者：
N. Rudin;E. Sugarman;J. Haber
中科院分区：
文献类型：
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作者：
N. Rudin;E. Sugarman;J. Haber
我们研究了HO内切酶诱导的酵母LACZ复制质粒中的双链断裂（DSB）重组和修复。一个117 bp的MATa片段，嵌入在一个拷贝的LACZ，作为一个网站的启动时，HO内切酶表达的DSB。DSB可以使用位于同一质粒上的LACZ的第二个无启动子拷贝上的野生型序列来修复。与正常的交配型转换相反，与基因转换相关的交换至少有50%的时间发生。当两个LACZ拷贝处于正方向时，伴随交换的转换事件的比例（80%）大于倒置时（50%）。此外，质粒丢失的分数在反向方向上显著更大。还监测了中间体和最终产物的出现动力学。DSB的修复是缓慢的，从检测到HO切割片段到完成修复至少需要一个小时。令人惊讶的是，两个互换的倒数产物的出现并不以相同的动力学发生。例如，当两个LACZ序列处于直接取向时，HO诱导的大环状缺失产物的形成并不伴随着小环状互补产物的出现。我们认为，这些差异可能反映了两个动力学上可分离的过程，一个只涉及一个切割端，另一个是由DSB两端的协同参与造成的。
We have investigated HO endonuclease-induced double-strand break (DSB) recombination and repair in a LACZ duplication plasmid in yeast. A 117-bp MATa fragment, embedded in one copy of LACZ, served as a site for initiation of a DSB when HO endonuclease was expressed. The DSB could be repaired using wild-type sequences located on a second, promoterless, copy of LACZ on the same plasmid. In contrast to normal mating-type switching, crossing-over associated with gene conversion occurred at least 50% of the time. The proportion of conversion events accompanied by exchange was greater when the two copies of LACZ were in direct orientation (80%), than when inverted (50%). In addition, the fraction of plasmids lost was significantly greater in the inverted orientation. The kinetics of appearance of intermediates and final products were also monitored. The repair of the DSB is slow, requiring at least an hour from the detection of the HO-cut fragments to completion of repair. Surprisingly, the appearance of the two reciprocal products of crossing over did not occur with the same kinetics. For example, when the two LACZ sequences were in the direct orientation, the HO-induced formation of a large circular deletion product was not accompanied by the appearance of a small circular reciprocal product. We suggest that these differences may reflect two kinetically separable processes, one involving only one cut end and the other resulting from the concerted participation of both ends of the DSB.