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复制起点的规范和调控。我们的模式生物，嗜热四膜虫，拥有一个21 kb的核糖体DNA（rDNA）微型染色体，在发育过程中扩增9,000倍，但在营养细胞分裂期间每个细胞周期复制一次。我们已经确定了反式作用因子，抑制或激活rDNA复制结合到一个共同的顺式作用复制决定簇，I型元件。我们最近发现，四膜虫起源识别复合物（ORC）包含一个完整的RNA亚基，形成沃森：克里克Dase与I型元件T-丰富的链对。值得注意的是，这种RNA，命名为26 T，对应于26 S核糖体RNA的3'末端。我们假设26 T RNA是从细胞质核糖体加工而来，并作为DNA复制和细胞代谢之间的生理联系发挥作用。我们进一步推测，RNA介导的识别仅限于rDNA复制子，并提供了一种机制，在程序化基因扩增过程中选择性地将ORC靶向rDNA来源。在本申请中，我们提出了实验来确定这种显着的ORC复合物的蛋白质组成。我们将研究26 TRNA残基对rDNA序列识别和起始激活的贡献，并将确定ORC如何识别缺乏26T.RNA结合位点的其他复制起始点。我们将测试26 T RNA生物发生模型，以深入了解调控，并将嵌合ARS 1/rDNA复制子引入发育中的大核，以检查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物化学和反向遗传方法，目的是阐明调节真核DNA复制起始的潜在机制。研究这种新的复制模式，RNA介导的起源识别，拟议的实验应该提供令人兴奋的新见解染色体生物学。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.