functional centromeres determine the activation time of pericentric origins of dna replication in saccharomyces cerevisiae着丝粒功能确定的激活时间pericentric在酿酒酵母dna复制的起源.pdfVIP

Functional Centromeres Determine the Activation Time of Pericentric Origins of DNA Replication in Saccharomyces cerevisiae 1,2 1,2 2 Thomas J. Pohl , Bonita J. Brewer , M. K. Raghuraman * 1 Molecular and Cellular Biology Program, University of Washington, Seattle, Washington, United States of America, 2 Department of Genome Sciences, University of Washington, Seattle, Washington, United States of America Abstract The centromeric regions of all Saccharomyces cerevisiae chromosomes are found in early replicating domains, a property conserved among centromeres in fungi and some higher eukaryotes. Surprisingly, little is known about the biological significance or the mechanism of early centromere replication; however, the extensive conservation suggests that it is important for chromosome maintenance. Do centromeres ensure their early replication by promoting early activation of nearby origins, or have they migrated over evolutionary time to reside in early replicating regions? In Candida albicans, a neocentromere contains an early firing origin, supporting the first hypothesis but not addressing whether the new origin is intrinsically early firing or whether the centromere influences replication time. Because the activation time of individual origins is not an intrinsic property of S. cerevisiae origins, but is influenced by surrounding sequences, we sought to test the hypothesis that centromeres influence replication time by moving a centromere to a late replication domain. We used a modified Meselson-Stahl density transfer assay to measure the kinetics of replication for regions of chromosome XIV in which either the functional centromere or a point-mutated version had been moved near origin