FISSION YEAST DNA REPLICATION ORGINS

描述：芽殖酵母S. 酿酒酵母是相对较好理解的。 这样的起源包含着一种约束力， 六个多肽来源识别复合物（ORC）和一个或 另外两种对其他因子的募集很重要的蛋白质 和/或DNA解旋。 ORC结合对于随后的 Cdc 6p、Mcm家族蛋白质以及可能的其他蛋白质的结合 G1阶段的开始。 在G1/S接口上，复制通过以下方式启动 一种未知的机制，伴随着Cdc 6p和Mcm的解离 家人 在植物中发现了ORC和Mcm蛋白的同源物， 动物和其他真菌，这表明启动的基本机制 在所有真核生物中都是保守的。 然而，其他起源 测试的生物体不如S中的明确。啤酒。 比如说， 相比S。酿酒酵母，有放松的复制特异性 起源于裂变酵母S.粟酒 此外，S.粟酒的起源是 比S大四倍多。酿酒，并有更多的 贡献序列元素。 拟议研究的目标是 鉴定与S.粟酒起源并将其与 S.啤酒。 这将使PI能够确定是否启动 在S.粟酒的起源需要新的蛋白质类型以及类似的蛋白质类型， 在S。啤酒。 为此，PI将使用连接器替换 诱变和体内足迹法来精确定义序列 对于两个独立的S。粟酒起源 蛋白质与 这些重要的序列也将通过遗传筛选和（在 与Jerard Hurwitz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.