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

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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