Mechanism of Initiation of Replication of the Chromosome VI of Saccharomyces cerevisiae

项目背景和目的：本项目是在世界上第一次发现、定位和鉴定真核生物单个染色体上的所有复制起点时启动的。本研究的目的是在酿酒酵母VI号染色体上发现9个起始点，以1）研究每个起始点的功能等级，2）确定每个起始点是否具有特定的调控作用，3）寻找起始点活性与细胞周期调控之间的特定联系。结果1）确定了所有起始点的起始频率，并将其分为3类：a）每个细胞周期使用一次的三个来源（高频来源），B）2-3个细胞周期使用一次的四个中频来源，c）2个低频来源使用少于5%。在S期同步进行过程中，确定了9个起始点的起始和复制时间，揭示了每个起始点都是在固定的时间起始的，从而导致顺序复制。 ...更多信息 从着丝粒到端粒的整个染色体的阳离子。而第一个起始点oriSC 607位于距离着丝粒43 kb处，并以双向方式复制。最新启动oriSC 609附近的端粒显示出独特的属性，作为启动和复制的时间的异质性。2)每个起始点的起始频率受参与复制前复合物形成和激活的基因突变的影响。特别是，第一个起始oriSC 607受大多数突变的影响最小，最后一个起始oriSC 609对激活基因的突变高度敏感，但受到复合物组分突变的刺激。3)我们已经发现，参与响应DNA损伤的S期进展的监视的基因Rad 53负控制晚期复制起点oriSC 609的起始。在rad 53突变体细胞中，没有发生对晚期起源起始的阻断，并且在DNA破坏剂MMS存在下复制继续，从而引起细胞死亡。这是第一次证明了在特定的生理条件下，如DNA损伤，多复制子在细胞周期进程中的生物调节作用。少

Background and Aim of the project.This project was initiated when we discovered, mapped and chracterized all replication origins on a single eukaryotic chromosome for the first time in the world. The aim of the project based on the finding of 9 origins on the chromosome VI of Saccharomyces cerevisiae was 1) to examine possible hierarchy in function of each origin, 2) to identify if any of specific regulation for each origin, 3) to find specific linkage between the origin activity and cell cycle regulation.Results 1) The frequency of initiation of all origins were determined to classify them into three groups : a) three origins used once in every cell cycle (high frequency origin), b) four intermediate frequency orihins used once in 2-3 cell cycle, c) 2low frequency origins used less than 5%. The timing of initiation and replication of 9 origins during synchronous progression of S-phase was determined to reveal that each origin is initiated at the fixed time resulting a sequential repli … More cation of the entire chromosome from centromere to telomere. However, the first initiating origin oriSC607 is located 43kb away from the centromere and replicated bidrectionally. The latest initiating oriSC609 near the telomere showed peculiar property as to the heterogeneity of timing of initiation and replication. 2) The frequency of initiation of each origin was variably affected by the mutation in genes involved in formation and activation of pre-replicative complex. In particular, the first initiating oriSC607 is least affected by most mutations and the last initiation oriSC609 is highly sensitive to mutations in activator genes, but rather stimulated by a mutaion in a component of the complex. 3) we have discovered that a gene Rad53 which ia involved in surveillance of S-phase progression in response to DNA damages negatively control the initiation of late replicating origin, oriSC609. In rad53 mutant cells, the blocking of the initiation of late origin did not occur and the replication continues in the presence a DNA demaging reagent MMS to give rise the cell death. This is the first demonstration of a biological of multi-replicons in the regulation of cell cycle progress in a specific physiological condition like the damage in DNA. Less