Contribution of Sperm Nucleus to Paternal DNA Replication

精子核对父本 DNA 复制的贡献

• 批准号: 8227938
• 负责人: WILLIAM S WARD
• 金额: $ 23.62万
• 依托单位: UNIVERSITY OF HAWAII AT MANOA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-19 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8227938
• 关键词: Base Pairing; Binding; Biological; CDC6 gene; CDT1 Gene; Cell Cycle; Cell Nucleus; Cell division; Cell physiology; Cells; Child; Chimeric Proteins; Chromatin; Clinical; Complex; Cytoplasm; DNA; DNA Sequence; DNA biosynthesis; DNA replication origin; DNA-Directed DNA Polymerase; Data; Elements; Embryo; Embryonic Development; Ensure; Epigenetic Process; Event; Fathers; Fertilization; Future; Genes; Genetic; Genetic Code; Genome; Germ Cells; Hour; Human; Human Genome; Immunofluorescence Immunologic; In Vitro; Injection of therapeutic agent; Instruction; Knowledge; Licensing; Life; MCM2 gene; Mammalian Cell; Mammals; Mediating; Messenger RNA; Modeling; Molecular; Mothers; Mus; Nuclear Matrix; ORC1L gene; Oocytes; Pathway interactions; Process; Protein Binding; Proteins; Publishing; Recruitment Activity; Regulation; Replication Licensing; Replication Origin; S Phase; Safety; Series; Site; Source; Structure; Testing; Time; Transgenic Mice; assisted reproduction; blastomere structure; egg; embryo cell; foot; human disease; inhibitor/antagonist; male; origin recognition complex; pressure; public health relevance; research study; sperm cell

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. PUBLIC HEALTH RELEVANCE: The 30,000 or so genes that make up the human genome physically reside in DNA, a long tape-like molecule. Each cell in the body has the entire DNA sequence, and it takes roughly 6 feet of DNA to encode all 30,000 genes. At fertilization, there are only two copies of this DNA, one from the mother and one from the father, and both need to be replicated flawlessly. This application proposes to test the idea that the sperm cell brings to the egg more than only the DNA, itself, but instructions on how to replicate it properly in the first embryonic cell division.

描述（由申请人提供）：哺乳动物的精子细胞提供了一个独特的机会，可以理解遗传密码以外的分子遗传原理，并且该知识直接与人类辅助生殖实践有关。紧凑型精子染色质几乎没有与正常细胞过程相关的所有酶促机制，并且仅保持介导遗传所需的表观遗传和遗传成分。我们建议在此应用中检验的假设是，除了父亲的DNA外，精子核提供了分子成分，这些分子成分对于适当起始和调节父亲DNA复制至关重要。没有这些元素，我们发布的数据表明，父亲DNA将永远不会被复制，并且不可能进行胚胎发育。意义：在这种增加的艺术时代，越来越多的人类胚胎会成为孩子的第一个，在临床操纵配子后，在体外定义了DNA合成。我们了解如何在配子存储期间保持DNA完整，但是我们不知道要保持适当的原点识别站点是必需的。由于哺乳动物的单细胞胚胎是了解细胞如何划分和许可DNA复制起源的特别合适模型，因此也有重要的生物学意义。 DNA复制机制也是许多人类疾病的重要目标。复杂过程的启动，控制和完成的分子机制，复制了60亿个乳腺DNA的碱基对。复制的每个来源都是通过由六种蛋白质ORC1L -ORC6L组成的原点识别复合物（ORC）开始的一系列蛋白质的“许可”。当许可的起源进入S相时，另一个蛋白质以特定方式辅助以最终募集DNA聚合酶。许可确保每个周期仅复制整个基因组的每个组成部分。在哺乳动物细胞中，ORC2L-5L在整个细胞周期中仍与复制起源约束，而ORC1L则募集到G1中的起源，并在S期间降解（通过细胞周期的ORC6L的命运是未知的）。在哺乳动物的一个细胞中，父亲和母体基因组是独立复制和异步复制的，并且我们的初步数据表明，在同一卵母细胞中，两种原始核的DNA合成的启动可以变化多达三个小时。在特定目标1中，我们将使用前核转移实验确定小鼠一个细胞胚胎中许可的时机。在特定目标2中，我们将测试我们假设的预测，即受精前已经存在于母体染色质上的ORC2L-5L，但是在受精后负载到雄性染色质。在特定目标3中，我们将使用ORC-GFP融合蛋白直接测试ORC2L-5L或ORC1L直接与精子核基质结合。未来的研究将确定ORC1L-5L蛋白结合的精子染色质中的表观遗传成分。 公共卫生相关性：构成人体基因组物理基因组的30,000个左右基因位于DNA，这是一种长胶带样分子。体内的每个细胞都有整个DNA序列，并且大约需要6英尺的DNA来编码所有30,000个基因。在受精时，只有两个副本的DNA副本，一个来自母亲，一个来自父亲的DNA，两者都需要完美地复制。该应用建议测试精子细胞将卵带入卵的想法不仅仅是DNA本身，但是指示如何在第一个胚胎细胞分裂中正确复制它。