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 origins that reside in a late replication region. We show that a functional centromere acts in cis over a distance as great as 19 kb to advance the initiation time of origins. Our results constitute a direct link between establishment of the kinetochore and the replication initiation machinery, and suggest that the proposed higher-order structure of the pericentric chromatin influences replication initiation.
Highlights
Centromere function, the ability to assemble a kinetochore and mediate chromosome segregation during meiosis and mitosis, is critical for the survival and propagation of eukaryotic organisms
We further show that centromere-mediated early origin activation depends on the centromere’s ability to recruit at least a subset of the proteins needed for chromosome segregation
To test the hypothesis that centromeres contribute to the early activation of these adjacent origins, we obtained a strain in which the centromere on chromosome XIV (Figure 1A) had been relocated to a distal location on the left chromosomal arm [27] (Figure 1B)
Summary
Centromere function, the ability to assemble a kinetochore and mediate chromosome segregation during meiosis and mitosis, is critical for the survival and propagation of eukaryotic organisms. It is important that properly functioning centromeres be established on both sister chromatids following replication of centromeric DNA and prior to the initiation of chromosome segregation. It has been hypothesized that early replication of centromere DNA provides sufficient time for the centromere-specific histone, CenH3, to be incorporated on both sister chromatids and to ensure subsequent microtubule attachment [14,15,16]. Consistent with this idea, Feng et al showed that a delay in centromere DNA replication in the absence of the replication checkpoint leads to increased aneuploidy [16]. It is thought that this observed increase in aneuploidy is due to the lack of properly bi-oriented sister chromatids
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