The control of Spo11's interaction with meiotic recombination hotspots
The control of Spo11's interaction with meiotic recombination hotspots
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DOI：
10.1101/gad.321105
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发表时间：
2005-01-15
影响因子：
10.5
通讯作者：
Klein, F
中科院分区：
文献类型：
--
作者：
Prieler, S;Penkner, A;Klein, F
程序性双链断裂(DSB)启动减数分裂重组，通过进化保守的拓扑异构酶同源物Spo11的活性产生。Spo11被认为在DSB形成的初始步骤中催化DNA裂解反应，而至少还有11个因子辅助酿酒酵母。利用染色质免疫沉淀法(CHIP)，我们检测了Spo11与野生型细胞减数分裂热点的瞬时、非共价关联。这种关联的建立需要Rec102、Rec104和Rec114，而能否及时将Spo11从染色质中移除取决于几个因素，包括Mei4和Ndt80。此外，至少还有一个组件，即Red1，负责将Spo11‘S互动局部限制在热点的核心区。在染色体铺展中，我们观察到减数分裂特异的Spo11-Myc焦点，从细线期到粗线期，与DSB的形成无关。在rad50S和com1Delta/sae2Delta突变体中，我们观察到了Spo1 I与热点之间的一种新的反应中间产物，这使得芯片可以在没有人工交联的情况下检测到全长的热点DNA。虽然这段DNA不含断裂，但它的回收需要Spo11的S催化残基Y135。我们认为，只有在Spo11切割反应的可逆阶段，即rad50S和com1Delta/sae2Delta突变体瞬间停止时，才有可能检测到未交联的全长热点DNA。
Pro-rammed double-strand breaks (DSBs), which initiate meiotic recombination, arise through the activity of the evolutionary conserved topoisomerase homolog Spo11. Spo11 is believed to catalyze the DNA cleavage reaction in the initial step of DSB formation, while at least a further 11 factors assist in Saccharomyces cerevisiae. Using chromatin-immunoprecipitation (ChIP), we detected the transient, noncovalent association of Spo11 with meiotic hotspots in wild-type cells. The establishment of this association requires Rec102, Rec104, and Rec114, while the timely removal of Spo11 from chromatin depends on several factors, including Mei4 and Ndt80. In addition, at least one further component, namely, Red1, is responsible for locally restricting Spo11's interaction to the core region of the hotspot. In chromosome spreads, we observed meiosis-specific Spo11-Myc foci, independent of DSB formation, from leptotene until pachytene. In both rad50S and com1Delta/sae2Delta mutants, we observed a novel reaction intermediate between Spo1 I and hotspots, which leads to the detection of full-length hotspot DNA by ChIP in the absence of artificial cross-linking. Although this DNA does not contain a break, its recovery requires Spo11's catalytic residue Y135. We propose that detection of uncross-linked full-length hotspot DNA is only possible during the reversible stage of the Spo11 cleavage reaction, in which rad50S and com1Delta/sae2Delta mutants transiently arrest.