FISSION YEAST DNA REPLICATION ORGINS

描述：芽殖酵母S. 酿酒人对它的理解相对较好。这样的起源包含一个绑定 六个多肽起源识别复合体(ORC)和一个或 另外两种对招募其他因子很重要的蛋白质 和/或用于解开DNA。ORC绑定似乎对于后续的 Cdc6p，MCM蛋白家族，可能还有其他蛋白在 G1期开始。在G1/S接口，复制通过以下方式启动 一种未知的机制，伴随着CDC6P和MCM的解离 一家人。在植物中已经发现了ORC和MCM蛋白的同源物， 动物和其他真菌，表明启动的基本机制 在所有真核生物中都是保守的。然而，起源于其他 被测试的生物体没有酿酒酵母中的定义那么明确。例如, 与酿酒酵母相比，复制有松散的专一性。 起源于分裂酵母S.pombe。此外，S.pombe的起源是 比酿酒酵母大四倍多，而且有更多 贡献序列元素。拟议研究的目标是 识别与S.pombe起源相互作用的蛋白质，并将它们与 在酿酒酵母中。这将使PI能够确定是否启动 在S.pombe起源需要新类型的蛋白质以及类似的蛋白质 在酿酒酵母中。为此，PI将使用链接器替换 突变和体内足迹以准确定义序列 对两个独立的S.pombe起源很重要。与之相互作用的蛋白质 这些重要的序列也将通过基因筛查和(在 与Jerard Hurwitz的合作)。

DESCRIPTION: The structure of replication origins in the budding yeast S. cerevisiae is relatively well understood. Such origins contain a binding site for the six polypeptide Origin Recognition Complex (ORC) and for one or two additional proteins important for the recruitment of other factors and/or for DNA unwinding. ORC binding appears necessary for the subsequent binding of Cdc6p, the Mcm family of proteins, and perhaps other proteins at the beginning of G1 phase. At the G1/S interface, replication initiates by an unknown mechanism, accompanied by dissociation of Cdc6p and the Mcm family. Homologs to the ORC and Mcm proteins have been found in plants, animals, and other fungi, suggesting that the basic mechanism of initiation is conserved in all eukaryotic organisms. Nevertheless, origins in other tested organisms are less well defined than in S. cerevisiae. For example, compared to S. cerevisiae, there is relaxed specificity for replication origins in the fission yeast S. pombe. In addition, S. pombe origins are more than four-fold larger than those of S. cerevisiae and have more contributing sequence elements. The goal of the proposed studies is to identify proteins that interact with S. pombe origins and compare them to those in S. cerevisiae. This will enable the PI to determine if initiation at S. pombe origins requires new types of proteins as well as ones similar to those in S. cerevisiae. To do this the PI will use linker substitution mutagenesis and in vivo footprinting to accurately define sequences important for two independent S. pombe origins. Proteins that interact with these important sequences also will be sought by genetic screening and (in collaboration with Jerard Hurwitz) by in vitro binding assays.