Molecular pathways of meiotic prophase checkpoints in mice

小鼠减数分裂前期检查点的分子途径

• 批准号: 467268969
• 负责人: Professor Dr. Attila Tóth
• 金额: --
• 依托单位: Institut für Physiologische Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/467268969?language=en
• 关键词: Molecular; pathways; meiotic; prophase; checkpoints

Halving of chromosome numbers in meiosis is the hallmark of the lifecycle of sexually-reproducing organisms. Meiotic segregation of chromosomes requires that homologous copies of each chromosome (homologs) form physical connections in meiotic prophase. In most taxa including mammals, these connections depend on chromosomal crossovers produced by recombination. Programmed formation of DNA double-strand breaks (DSBs) initiates meiotic recombination. Meiotic DSBs result in single stranded DNA (ssDNA) ends which invade homologs promoting chromosomal synapsis. In turn, synapsis promotes recombination-mediated DSB repair and generation of crossovers.To protect genome integrity in meiosis, quality control mechanisms/checkpoints ensure that all homolog pairs synapse, and that DSBs, which are potentially genotoxic, are repaired before meiocytes exit prophase. A central unresolved question is whether the DNA-damage checkpoint carries out the quality control for both DSB repair and synapsis or if distinct checkpoints monitor DSB repair and synapsis. This question is rooted in a debate on the role of HORMAD2, which we identified as a critical component in meiosis-specific quality control of recombination. HORMAD2 preferentially binds to chromosomal regions where synapsis has not formed yet. Unrepaired DSBs are also concentrated in these regions.Based on our data we hypothesize that HORMAD2 recruits ATR to unsynapsed chromosomal regions, and establishes a synapsis checkpoint that does not require DSBs to trigger elimination of synapsis-deficient meiocytes. In an alternative hypothesis, HORMAD2 blocks repair of DSBs to activate a DNA-damage checkpoint in unsynapsed regions instead of, or in addition to, a synapsis checkpoint. Verdict on these two models was hindered by three main factors. First, proper meiotic DSB repair and chromosomal synapsis are mutually dependent, making it difficult to separate checkpoint activation by defects in synapsis and/or DSB repair. Second, it is unclear if DSB-independent binding of HORMAD2-ATR complex to unsynapsed regions can establish a checkpoint. Third, standard methods, which rely on marker proteins to monitor DSB repair, failed to conclusively answer if HORMAD2 blocks DSB repair until proper synapsis is formed.We developed two new mouse models and a new method which overcome the main impediments in meiotic checkpoint analysis: (1) a mouse model that separates DSB repair and synapsis defects (factor 1), (2) a mouse model where HORMAD2 fails to bind chromosomes (factor 2), and (3) a method for the direct detection of meiotic ssDNA ends instead of protein markers of recombination (factor 3). Using these tools we will decisively test current models for meiosis quality control, revealing fundamental features of genetic inheritance with implication for human reproduction health.

减数分裂中染色体数目减半是有性生殖生物生命周期的标志。染色体的减数分裂分离要求每个染色体的同源拷贝（同源物）在减数分裂前期形成物理连接。在包括哺乳动物在内的大多数分类群中，这些联系依赖于重组产生的染色体交换。DNA双链断裂（DSB）的程序性形成启动减数分裂重组。减数分裂DSB产生单链DNA（ssDNA）末端，其侵入促进染色体联会的同源物。为了在减数分裂中保护基因组的完整性，质量控制机制/检查点确保所有同源物对突触，并确保具有潜在遗传毒性的DSB在性母细胞退出前期之前被修复。一个中心的未解决的问题是DNA损伤检查点是否进行DSB修复和突触的质量控制，或者是否有不同的检查点监测DSB修复和突触。这个问题是植根于辩论的作用HORMAD 2，我们确定为一个关键组成部分减数分裂特异性重组的质量控制。HORMAD 2优先结合到突触尚未形成的染色体区域。未修复的DSB也集中在这些区域。基于我们的数据，我们假设HORMAD 2招募ATR到未突触的染色体区域，并建立一个突触检查点，不需要DSB触发突触缺陷性母细胞的消除。在另一种假设中，HORMAD 2阻断DSB的修复，以激活非突触区域中的DNA损伤检查点，而不是突触检查点，或除了突触检查点之外。对这两种模式的判断受到三个主要因素的阻碍。首先，正确的减数分裂DSB修复和染色体突触是相互依赖的，使得难以通过突触和/或DSB修复中的缺陷来分离检查点激活。其次，目前尚不清楚HORMAD 2-ATR复合物与非突触区域的DSB非依赖性结合是否可以建立检查点。第三，依赖于标记蛋白来监测DSB修复的标准方法未能最终回答HORMAD 2是否阻断DSB修复直到形成适当的突触。我们开发了两种新的小鼠模型和一种新方法，克服了减数分裂检查点分析的主要障碍：（1）分离DSB修复和突触缺陷的小鼠模型（因子1），（2）HORMAD 2不能结合染色体的小鼠模型（因子2），和（3）直接检测减数分裂ssDNA末端而不是重组的蛋白质标记物的方法（因子3）。使用这些工具，我们将果断地测试当前的减数分裂质量控制模型，揭示遗传遗传的基本特征，并对人类生殖健康产生影响。