Eukaryotic Chromosome Replication

描述（由申请人提供）：真核生物染色体的复制是一个有序的过程，涉及在S期激活多组复制起点。然而，在S阶段，并非所有的起源都同时着火。正是这种在S期（起源激活的时间程序）中调控的起源激活一直让我们着迷，我们建议继续在出芽酵母Saccharomyces cerevisiae中研究。提出的工作包括以下四个方面：(1)Cdk1的S期激活剂，Clb5p和Clb6p，在起始放电方面是不相等的。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作用在迟发起源方面的机制差异模型。我们还将探讨CDK与另一种S期激酶Cdc7p之间的相互作用。早期和晚期起源的区别尚不清楚。我们想象有一个S相时钟，在S相的不同时间为不同的起始点提供关键的起始因子。文献报道表明，用羟基脲（HU）处理的细胞或RAD53突变的细胞在晚起源方面显示出改变的时钟。我们建议探索核苷酸池和S期检查点蛋白Rad53p在S期时钟中的作用。当一个起源在S期激活时，在很大程度上是由于该起源的染色体背景。端粒附近的起源是晚期复制；着丝粒附近的起源是早期复制。我们希望测试染色体结构域之间的这种时间区分对染色体维持和分离很重要的想法。(4)负责原点计时的顺式调控要素的定义很模糊。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，以期定义顺式调控序列并阐明其作用机制。

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.