Molecular structures and mechanisms of DNA break processing in mouse meiosis. Molecular structures and mechanisms of DNA break processing in mouse meiosis

Exonucleolytic resection is a critical step in repair of DNA double-strand breaks (DSBs) via homologous recombination, but resection mechanisms are not well understood, particularly in mammalian meiosis. Here, we use a genome-wide, nucleotide-resolution analysis to define the molecular structure of resected DSBs in mouse spermatocytes. Resection tracts averaged 1100 nucleotides on each side of a DSB, but with a broad distribution and substantial fine-scale heterogeneity at individual hotspots. Surprisingly, eliminating the nuclease activity of EXO1 only modestly decreased resection lengths, thus EXO1 is not the major 5′→3′ exonuclease in mouse meiosis. In contrast, the DSB-responsive kinase ATM proved to be a key regulator of both the initiation and extension of resection. In wild type, apparent intermolecular recombination intermediates clustered near to but offset from DSB positions, consistent with joint molecules with incompletely invaded 3′ ends. Finally, we provide evidence for PRDM9-dependent chromatin remodeling leading to increased accessibility at recombination sites. Our findings give insight into the mechanisms of DSB processing and repair in meiotic chromatin. Overall design: Detection of double-strand break end resection and changes in local chromatin structure at recombination sites in mouse meiosis

核酸外切切除（Exonucleolytic resection）是通过同源重组修复DNA双链断裂（DSBs）的关键步骤，但目前对其切除机制尚不清楚，尤其是在哺乳动物减数分裂过程中。本研究借助全基因组核苷酸分辨率分析，解析了小鼠精母细胞中经切除的DSBs的分子结构。DSB两侧的切除区段平均长度为1100个核苷酸，但分布范围宽泛，且在单个重组热点处存在显著的精细尺度异质性。令人意外的是，敲除EXO1的核酸酶活性仅会轻度缩短切除区段，这表明EXO1并非小鼠减数分裂中主要的5′→3′核酸外切酶。与之相反，响应DNA双链断裂的激酶ATM被证实是切除起始与延伸过程的关键调控因子。在野生型样本中，表观可见的分子间重组中间产物聚集于DSB位点附近但存在位置偏移，这与带有未完全侵入3′端的联结分子特征相符。最后，本研究为依赖PRDM9的染色质重塑可提升重组位点处染色质可及性提供了实验证据。我们的研究结果为阐明减数分裂染色质中DSB加工与修复的分子机制提供了新见解。实验整体设计：检测小鼠减数分裂重组位点处的双链断裂末端切除情况及局部染色质结构变化。