Eukaryotic Chromosome Replication

描述(申请人提供)：真核细胞染色体的复制是一个有序的过程，在S阶段涉及多组复制起点的激活。然而，在S阶段，并不是所有的起源都同时发生。正是S阶段(起源激活的时间程序)中起源的这种受调控的激发一直吸引着我们，我们建议继续在萌芽酵母中进行研究。拟做的四个方面的工作包括：(1)CdK_1、ClB_5P和ClB_6P的S相激活剂在起火方面不等价。与Clb5p不同，Clb6p在激活晚期起源方面效率低下。我们将测试Clb5p和Clb6p在延迟发射起源方面的作用机制差异的模型。我们还将探讨CDK与另一种S相激酶--CDC7P之间的相互作用。(2)早发火和晚发火起源的区别尚不清楚。我们设想有一个S相钟，它在S相中的不同时间向不同的起源发出关键的启动因素。文献中的报告表明，羟基脲(HU)处理的细胞或在RAD53中突变的细胞显示出关于晚期起源的时钟改变。我们建议探索核苷酸池和S相检查点蛋白Rad53p在S相钟中的作用。(3)当一个起源在S阶段发生时，很大程度上是由于该起源的染色体背景。端粒附近的起始点复制较晚；着丝粒附近的起始点复制较早。我们希望测试染色体区域之间的这种时间差异对于染色体维护和分离是重要的这一想法。(4)只负责起源时间的顺式调控元件被模糊地定义。我们建议扩展我们的全基因组复制研究，将晚期起源描述为一类，希望定义顺式调控序列并阐明它们的作用机制(S)。

DESCRIPTION (provided by applicant): Replication of eukaryotic chromosomes is an ordered process involving the activation of multiple sets of replication origins during S phase. Within S phase, however, not all origins fire at the same time. It is this regulated firing of origins within S phase (the temporal program of origin activation) that has long fascinated us and that we propose to continue to study in budding yeast Saccharomyces cerevisiae. The four areas of proposed work include the following: (1) The S phase activators of Cdk1, Clb5p and Clb6p, are not equivalent with respect to origin firing. Clb6p, unlike Clb5p, is inefficient at activating late origins. We will test models for the mechanistic differences between Clb5p and Clb6p action with regard to late-firing origins. We will also explore the interaction between CDK and the other S phase kinase, Cdc7p. (2) What distinguishes early- from late-firing origins is unknown. We imagine there is an S phase clock that metes out critical initiation factors to different origins at different times during S phase. Reports in the literature suggest that cells treated with hydroxyurea (HU) or mutated in RAD53 show an altered clock with regard to late origins. We propose to explore the role of nucleotide pools and the S-phase checkpoint protein Rad53p in the S phase clock. (3) When an origin fires in S phase is due in large part to the chromosomal context of that origin. Origins near telomeres are late replicating; origins near centromeres are early replicating. We wish to test the idea that this temporal distinction between chromosome domains is important for chromosome maintenance and segregation. (4) The cis-regulatory elements responsible for origin timing only have been vaguely defined. We propose to extend our whole-genome replication studies to characterize late origins as a class in hopes of defining the cis-regulatory sequences and elucidating their mechanism(s) of action.