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

项目的背景和目的。当我们在世界上第一次发现，绘制和描述了单个真核生物染色体上所有复制起源时，这个项目就开始了。基于对酿酒酵母6号染色体上9个起源的发现，本项目的目的是：1)检查每个起源在功能上可能的层次结构；2)确定每个起源是否有任何特定的调控；3)发现起源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，将其分为3组：a)每个细胞周期使用一次的3个起始点（高频起始点），b) 2-3个细胞周期使用一次的4个中频起始点，c)使用不到5%的20个低频起始点。在s期的同步进程中，对9个起始点的起始和复制时间进行了测定，结果表明每个起始点都在固定时间启动，从而导致从着丝粒到端粒的整个染色体的阳离子增多。然而，第一个起始点oriSC607位于离着丝粒43kb的地方，并且是双向复制的。最近在端粒附近起始的oriSC609在起始时间和复制时间上表现出独特的异质性。2)参与复制前复合体形成和激活的基因突变对每个起源的起始频率有不同的影响。特别是，第一个启动oriSC607受大多数突变的影响最小，而最后一个启动oriSC609对激活基因的突变高度敏感，而是由复合物组分的突变刺激。3)我们发现了一个基因Rad53，它参与了对DNA损伤响应的s期进展的监视，负向控制晚期复制起源的起始，oriSC609。在rad53突变细胞中，延迟起始的阻断没有发生，在DNA破坏试剂MMS存在的情况下，复制继续进行，导致细胞死亡。这是第一次证明在特定的生理条件下，如DNA损伤，多复制子调控细胞周期进程。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