In vitro reconstitution of meiotic DNA double-strand-break formation

》》文章原文链接：In vitro reconstitution of meiotic DNA double-strand-break formation

》》Journal：Nature

》》相关产品：Protease Inhibitor Cocktail (EDTA-Free,100× in DMSO) (SJ-MK0001)

》》产品引用描述：

》》Abstract：

The Spo11 complex catalyses the formation of DNA double-strand breaks (DSBs), initiating meiotic recombination-a process that is essential for fertility and genetic diversity^1,2. Although the function of Spo11 has been known for 27 years, previous efforts to reconstitute DSB formation in vitro have been unsuccessful. Here we biochemically characterize the mouse SPO11-TOP6BL protein complex, and show that this complex cleaves DNA and covalently attaches to the 5' terminus of DNA breaks in vitro. Using a point-mutation strategy, we reveal that Mg²⁺ is essential for the DNA-cleavage activity of this complex in vitro, as confirmed by knock-in mice carrying a point mutation in SPO11 that disrupts its binding to Mg²⁺, thereby abolishing DSB formation. However, the activity of the SPO11 complex is ATP-independent. We also present evidence that the mouse SPO11 complex is biochemically distinct from the ancestral topoisomerase VI. Our findings establish a mechanistic framework for understanding the first steps of meiotic recombination.

》》部分实验数据展示：

Fig. 1 Biochemical characterization of the SPO11–TOP6BL complex. d, Analysis of DSS-cross-linked samples by SDS–PAGE (left) and

western blot using anti-His (middle) and anti-Flag (right) antibodies to detect His–SPO11 and TOP6BL–Flag, respectively (n = 3 independent experiments). CBB, Coomassie brilliant blue. g, Co-immunoprecipitation analysis showing Flag-immunoprecipitation (Flag-IP) detection of TOP6BL–Flag, His–SPO11, His–MBP–SPO11 (anti-His) and TOP6BL–tStrep (anti-StrepII) (n = 3 independent experiments).