Reconstitution of SPO11-dependent double-strand break formation

Homologous meiotic recombination starts with DNA double-strand breaks (DSBs) generated by SPO11 protein. SPO11 is critical for meiosis in most species but the DSBs it makes are also dangerous because of their mutagenic and gametocidal potential, so cells must foster beneficial functions of SPO11 while minimizing its risks. SPO11 mechanism and regulation remain poorly understood. Here we report reconstitution of DNA cleavage in vitro with purified recombinant mouse SPO11 bound to its essential partner TOP6BL. Similar to their yeast orthologs, SPO11–TOP6BL complexes are monomeric (1:1) in solution and bind tightly to DNA. Unlike in yeast, however, dimeric (2:2) assemblies of mouse SPO11–TOP6BL cleaves DNA to form covalent 5 prime attachments requiring SPO11 active site residues, divalent metal ions, and SPO11 dimerization. Surprisingly, SPO11 can also manifest topoisomerase activity by relaxing supercoils and resealing DNA that it has nicked. Structure modeling with AlphaFold3 illuminates the protein-DNA interface and suggests that DNA is bent prior to cleavage. Deep sequencing of in vitro cleavage products reveals a rotationally symmetric base composition bias that partially explains DSB site preferences in vivo. Cleavage is inefficient on complex DNA substrates, partly because SPO11 is readily trapped in DSB-incompetent (presumably monomeric) binding states that exchange slowly. However, cleavage is improved by using substrates that favor DSB-competent dimer assembly, or by fusing SPO11 to an artificial dimerization module. Our results inform a model in which intrinsically feeble dimerization restrains SPO11 activity in vivo, making it exquisitely dependent on accessory proteins that focus and control DSB formation so that it happens only at the right time and the right places.同源减数分裂重组起始于SPO11蛋白产生的DNA双链断裂（double-strand breaks, DSBs）。SPO11对多数物种的减数分裂过程至关重要，但其诱导产生的DSBs却因具有诱变和杀配子潜能而存在安全风险，因此细胞必须在保留SPO11有益功能的同时，最大限度降低其潜在危害。目前人们对SPO11的作用机制与调控方式仍知之甚少。本研究通过纯化与必需伴侣蛋白TOP6BL结合的重组小鼠SPO11，在体外重建了DNA切割反应体系。与酵母同源物类似，小鼠SPO11–TOP6BL复合物在溶液中以单体（1:1比例）形式存在，并可与DNA紧密结合。然而与酵母不同的是，小鼠SPO11–TOP6BL的二聚体（2:2比例）组装体可切割DNA，形成共价结合的5'端附着产物，该过程依赖SPO11的活性位点残基、二价金属离子以及SPO11的二聚化作用。令人意外的是，SPO11还可表现出拓扑异构酶活性：通过松弛DNA超螺旋并重新连接其切割产生的DNA切口。通过AlphaFold3进行的结构建模清晰揭示了蛋白质-DNA互作界面，并提示DNA在切割前会发生弯曲。对体外切割产物的深度测序结果显示，其碱基组成存在旋转对称性偏好，这一现象可部分解释体内DSB的位点选择偏好性。在复杂DNA底物上的切割效率较低，部分原因是SPO11易被困于无法介导DSB形成的结合状态（推测为单体状态），且此类状态的交换速率较慢。不过，通过使用更利于形成可介导DSB的二聚体组装体的底物，或将SPO11与人工二聚化模块融合，可提升切割效率。本研究结果支持如下模型：SPO11本身的二聚化能力较弱，这一特性在体内会抑制其活性，使其严格依赖辅助蛋白来聚焦并调控DSB的形成，从而确保DSB仅在正确的时间和位置发生。