Germline mutagenesis at meiotic double-strand breaks

项目概要/摘要 减数分裂重组对于哺乳动物生殖细胞的减数细胞分裂至关重要，因此对于 单倍体配子（即精子和卵子）的发育。重组是由数百个 DNA 双链启动的 由 SPO11 蛋白催化的全基因组链断裂 (DSB)。忠实传输 基因组传递给后代需要对这些众多的 DSB 进行适当的修复，主要是通过 与同系物重组。 DSB 的形成在减数分裂细胞中由 ATM 激酶调节，ATM 激酶是 已知是有丝分裂细胞中 DSB 的主要反应者，因此在缺乏 ATM 的情况下，减数分裂 DSB 增加~10倍。 我们最近发现减数分裂 DSB 有引发种系重排的风险， 涉及非同源末端连接的特定删除和串联重复，尤其是在缺乏 自动提款机。这些事件对于破坏这些热点发生的基因来说也是重要的 作为控制这些位点重组的相关 PRDM9 结合位点。因此，我们的研究结果揭示了 先前隐藏的种系突变潜力可能会影响人类健康和基因组 进化。在人类中，最近对冰岛人进行的远程测序支持了这种影响。本提案追求 旨在了解引起这些事件的机制、减数分裂 DSB 的事件范围，以及 年龄的影响。我们假设减数分裂 DSB 上可能存在其他重排，而不是我们现有的重排 先前已确定。因此，在第一个目标中，我们建议确定可以的诱变结果的范围 由减数分裂 DSB 产生，包括长程缺失和重复以及染色体易位。在 第二个目标是，我们研究可能影响缺失形成的因素。我们专注于DNA的作用 末端处理分两个步骤，SPO11去除和末端处理以及重组。此外，我们解决 附近 DSB 处形成的间隙是否是同源重组的底物以及父系的影响 减数分裂 DSB 重排事件中的年龄。

Project Summary/Abstract Meiotic recombination is essential for the reductional cell division in mammalian germ cells and thus for the development of haploid gametes, i.e., sperm and eggs. Recombination is initiated by hundreds of DNA double- strand breaks (DSBs) introduced genome-wide that are catalyzed by the SPO11 protein. Faithful transmission of the genome to subsequent generations requires proper repair of these numerous DSBs, primarily through recombination with the homolog. DSB formation is regulated in meiotic cells by the ATM kinase, which is known to be a primary responder to DSBs in mitotic cells, such that in the absence of ATM, meiotic DSBs increase ~10-fold. We recently discovered that meiotic DSBs are at risk for provoking germline rearrangements, in particular deletions and tandem duplications involving nonhomologous end-joining, especially in the absence of ATM. These events are consequential in terms of disrupting the genes in which these hotspots occur as well as the associated PRDM9 binding sites that govern recombination at those loci. Thus, our findings reveal a previously hidden potential for germline mutagenesis that is likely to affect human health and genome evolution. In humans, recent long-range sequencing of Icelanders supports this impact. This proposal pursues aims to understand the mechanisms that give rise to these events, the range of events at meiotic DSBs, and the effect of age. We hypothesize that other rearrangements are possible at meiotic DSBs than what we have previously identified. Thus, in the first aim, we propose to determine the range of mutagenic outcomes that can arise from meiotic DSBs, including long-range deletions and duplications and chromosomal translocations. In the second aim, we examine factors that may impact the formation of deletions. We focus on the effect of DNA end processing at two steps, SPO11 removal and end processing, and recombination. Further, we address whether gaps formed at nearby DSBs are substrates for homologous recombination and the impact of paternal age in the rearrangement events at meiotic DSBs.