Replication and Amplification of Tetrahymena rDNA

描述（由申请人提供）：本提案的目标是深入了解真核生物DNA复制起源的规范和调控。我们的模式生物，嗜热四膜虫，拥有一条21 kb的核糖体DNA （rDNA）小染色体，在发育过程中扩增9000倍，但在营养细胞分裂的每个细胞周期中复制一次。我们已经确定了通过结合常见的顺式作用复制决定因素（I型元件）来抑制或激活rDNA复制的反式作用因子。我们最近发现，四膜虫起源识别复合体（ORC）含有一个完整的RNA亚基，它与I型元素富t链形成沃森：克里克碱基对。值得注意的是，该RNA被命名为26T，对应于26S核糖体RNA的极端3'端。我们假设26T RNA是由细胞质核糖体加工而成，并作为DNA复制和细胞代谢之间的生理联系。我们进一步推测，rna介导的识别仅限于rDNA复制子，并提供了一种在程序化基因扩增过程中选择性地将ORC靶向rDNA起源的机制。在这个应用中，我们提出了实验来确定这个显著的ORC复合物的蛋白质组成。我们将研究26T RNA残基对rDNA序列识别和起源激活的贡献，并将确定ORC如何识别缺乏26T RNA结合位点的其他复制起源。我们将测试26T RNA生物发生模型以深入了解调控，并将嵌合的ARS1/rDNA复制子引入发育中的巨核以研究程序化基因扩增的分子基础。对于这些研究，我们将采用强大的生化和反向遗传方法，以阐明调节真核生物DNA复制起始的潜在机制。研究这种新的复制模式的实验，rna介导的起源识别，应该为染色体生物学提供令人兴奋的新见解。核糖体RNA在DNA复制中的作用这一前所未有的发现表明，这些研究将在复制领域内外产生广泛的影响。

DESCRIPTION (provided by applicant): The goal of this proposal is to gain insight into the specification and regulation of eukaryotic origins of DNA replication. Our model organism, Tetrahymena thermophila, harbors a 21 kb ribosomal DNA (rDNA) minichromosome that is amplified 9,000-fold during development, but is replicated once per cell cycle during vegetative cell divisions. We have identified trans-acting factors that repress or activate rDNA replication by binding to a common cis-acting replication determinant, the type I element. We recently discovered that the Tetrahymena Origin Recognition Complex (ORC) contains an integral RNA subunit that forms Watson:Crick Dase pairs with the type I element T-rich strand. Remarkably this RNA, designated 26T, corresponds to the extreme 3' terminus of 26S ribosomal RNA. We hypothesize that 26T RNA is processed from cytoplasmic ribosomes and functions as a physiological link between DNA replication and cellular metabolism. We further speculate that RNA-mediated recognition is restricted to the rDNA replicon and provides a mechanism for selectively targeting ORC to the rDNA origin during programmed gene amplification. In this application, we propose experiments to determine the protein composition of this remarkable ORC complex. We will examine the contribution 26T RNA residues to rDNA sequence recognition and origin activation, and will determine how ORC recognizes other replication origins that lack binding sites for 26T.RNA. We will test models for 26T RNA biogenesis to gain insights into regulation, and will introduce chimeric ARS1/rDNA replicons into the developing macronucleus to examine the molecular basis for programmed gene amplification. For these studies, we will employ powerful biochemical and reverse genetic approaches, with the goal of elucidating underlying mechanisms that regulate the initiation of eukaryotic DNA replication. The proposed experiments for studying this new replication paradigm, RNA-mediated origin recognition, should provide exciting new insights into chromosome biology. The unprecedented discovery of a role for ribosomal RNA in DNA replication indicates that these studies will have broad implications in and beyond the replication field.