Contribution of Sperm Nucleus to Paternal DNA Replication

精子核对父本 DNA 复制的贡献

• 批准号: 7884659
• 负责人: WILLIAM S WARD
• 金额: $ 24.61万
• 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/7884659
• 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; Mammalian Cell; Mammals; Mediating; Messenger RNA; Modeling; Molecular; Mothers; Mus; Nuclear Matrix; ORC1L gene; Oocytes; Pathway interactions; Phase; Process; Protein Binding; Proteins; Publishing; Recruitment Activity; Regulation; Replication Licensing; Replication Origin; 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）：哺乳动物精子细胞提供了一个独特的机会来理解遗传密码之外的分子遗传原理，这些知识对人类辅助生殖实践有直接的影响。致密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，只维持介导遗传所必需的表观遗传和遗传成分。我们提出在这个应用中测试的假设是，除了父亲的DNA外，精子核还提供了对正确启动和调节父亲DNA复制至关重要的分子成分。没有这些元素，我们发表的数据表明，父系DNA永远不会被复制，胚胎发育也不可能。意义：在这个抗逆转录病毒技术日益普及的时代，越来越多将成为儿童的人类胚胎在临床操作配子后，在体外进行这第一轮、决定性的DNA合成。我们知道如何在配子存储过程中保持DNA的完整，但我们不知道维持适当的起源识别位点所需的参数。这也有重要的生物学意义，因为哺乳动物单细胞胚胎是理解细胞如何划分和许可DNA复制起源的特别合适的模型。DNA复制机制也是许多人类疾病的重要靶点。哺乳动物DNA 60亿个碱基对的复杂复制过程的开始、控制和完成的分子机制现在已经被很好地理解了。每个复制起点都是通过一系列蛋白质的结合获得“许可”的，从由6个蛋白质组成的起点识别复合体（ORC）开始，ORC1L - ORC6L。当许可的起源进入s期时，另一宿主蛋白质以特定的方式结合，最终招募DNA聚合酶。许可确保整个基因组的每个组成部分每个周期只复制一次。在哺乳动物细胞中，ORC2L-5L在整个细胞周期中都与复制起点结合，而ORC1L在G1期被招募到起点，并在s期被降解（ORC6L在细胞周期中的命运尚不清楚）。在哺乳动物的一个细胞中，父本和母本基因组是独立和异步复制的，我们的初步数据表明，在同一个卵母细胞质中，两个原核的DNA合成起始时间可能相差3个小时。在特异性目标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，我们将验证我们的假设预测，即ORC2L-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Specific Aim 3中，我们将使用ORC-GFP融合蛋白测试ORC2L-5L或ORC1L是否直接与精子核基质结合。未来的研究将确定精子染色质中与ORC1L-5L蛋白结合的表观遗传成分。