Contribution of Sperm Nucleus to Paternal DNA Replication

描述（由申请人提供）：哺乳动物精子细胞提供了一个独特的机会，以了解遗传密码以外的分子遗传原理，这些知识对人类辅助生殖实践有直接影响。紧凑的精子染色质几乎没有与正常细胞过程相关的所有酶机制，并且仅维持介导遗传所必需的表观遗传和遗传组分。我们在本申请中提出的假设是，除了父亲的DNA之外，精子核还提供了对父亲DNA复制的适当启动和调节至关重要的分子组分。如果没有这些元素，我们发表的数据表明，父亲的DNA将永远不会被复制，胚胎发育将不可能。重要性：在这个ART增加的时代，越来越多的人类胚胎将成为儿童，在临床操作配子后，首先进行体外DNA合成。我们知道如何在配子储存过程中保持DNA的完整性，但我们不知道什么参数是必要的，以保持适当的起源识别位点。还有重要的生物学意义，因为哺乳动物单细胞胚胎是一个特别合适的模型，用于了解细胞如何划分和许可DNA复制起点。DNA复制机制也是许多人类疾病的重要靶点。哺乳动物DNA的60亿个碱基对复制的复杂过程的启动、控制和完成的分子机制现在已经很清楚了。每个复制起点都是通过一系列蛋白质的结合来“许可”的，从由六种蛋白质组成的起点识别复合物（ORC）开始，即ORC 1 L-ORC 6L。当许可的起源进入S期时，另一个蛋白质宿主以特定的方式缔合，最终招募DNA聚合酶。许可证确保整个基因组的每个组成部分在每个周期只复制一次。在哺乳动物细胞中，ORC 2L-5L在整个细胞周期中保持与复制起点结合，而ORC 1 L在G1中被募集到起点，并在S期降解（ORC 6L在整个细胞周期中的命运未知）。在哺乳动物的一个细胞中，父亲和母亲的基因组是独立和异步复制的，我们的初步数据表明，在同一个卵母细胞的细胞质中，两个原核的DNA合成的起始可以变化多达三个小时。在具体目标1中，我们将使用原核移植实验确定小鼠单细胞胚胎的许可时间。在具体目标2中，我们将测试我们假设的预测，即ORC 2L-5L在受精前已经存在于母体染色质上，但在受精后加载到雄性染色质上。在特定目标3中，我们将使用ORC-GFP融合蛋白测试ORC 2L-5L或ORC 1 L是否直接结合精子核基质。未来的研究将确定精子染色质中与ORC 1 L-5L蛋白结合的表观遗传成分。

DESCRIPTION (provided by applicant): The mammalian sperm cell offers a unique opportunity to understand the principles of molecular inheritance beyond the genetic code, and this knowledge has a direct bearing on human assisted reproduction practices. The compact sperm chromatin is devoid of virtually all enzymatic machinery associated with normal cellular processes, and only maintains the epigenetic and genetic components that are necessary to mediate inheritance. The hypothesis which we propose to test in this application is that in addition to the father's DNA the sperm nucleus provides molecular components that are essential for the proper initiation and regulation of paternal DNA replication. Without these elements, our published data suggest that the paternal DNA would never be replicated, and embryonic development would not be possible. Significance: In this era of increased ART, more and more human embryos that will become children undergo this first, defining round of DNA synthesis in vitro after clinical manipulation of the gametes. We understand how to keep the DNA intact during gamete storage, but we do not know what parameters are necessary to maintain proper origin recognition sites. There are also important biological implications as the mammalian one-cell embryo is a particularly suitable model for understanding how cells demarcate and license DNA replication origins. DNA replication mechanisms are also important targets for many human diseases. The molecular machinery for the initiation, control, and completion of the complicated process by which the 6 billion base pairs of mammalian DNA is replicated is now well understood. Each origin of replication is "licensed" by the binding of series of proteins, beginning with the origin recognition complex (ORC) made up of six proteins, ORC1L - ORC6L. When licensed origins enter S-phase, another host of proteins associate in a specific manner to eventually recruit DNA polymerases. Licensing ensures that each component of the entire genome is replicated only once per cycle. In mammalian cells ORC2L-5L remain bound to replication origins throughout the cell cycle, while ORC1L is recruited to the origin in G1, and degraded during S-phase (the fate of ORC6L through the cell cycle is unknown). In the mammalian one cell the paternal and maternal genomes are replicated independently, and asynchronously, and our preliminary data suggest that initiation of DNA synthesis of two pronuclei in the same oocyte cytoplasm can vary by as much as three hours. In Specific Aim 1 we will determine the timing of licensing in the mouse one cell embryo using pronuclear transfer experiments. In Specific Aim 2 we will test the prediction of our hypothesis that ORC2L-5L are already present on the maternal chromatin before fertilization, but load onto the male chromatin after fertilization. In Specific Aim 3 we will test whether ORC2L-5L or ORC1L binds directly to the sperm nuclear matrix, using ORC-GFP fusion proteins. Future studies will identify the epigenetic component in sperm chromatin to which ORC1L-5L proteins bind.