Global control of DNA replication timing by the budding yeast telomere protein Rif1

摘要

BackgroundDNA replication in eukaryotes is initiated from specificchromosomal sites (origins) that fire in a defined, celltype-specific temporal pattern. This replication programappears to be under epigenetic control through mechanismsthat are still poorly understood.Materials and methodsStudies were done in Saccharomyces cerevisiae (W303background) using standard genetic and molecularmethods. Chromatin immunoprecipitation assays wereperformed in strains in which genes encoding DNApolymerase 1 or 2 were epitope-tagged at their endogenousloci.ResultsWe showed previously that telomere TG-repeat tractlength exerts an epigenetic effect in cis on the activity ofnearby subtelomeric replication origins, such that ashortened telomere will replicate earlier [1]. Here weshow that deletion of the RIF1 gene, which encodes atelomere-specific Rap1-interacting protein involved intelomere length regulation and telomere “capping” [2-5],also leads to premature replication of two different subtelomericregions examined. A similar effect of RIF1deletion on other subtelomeric regions has recentlybeen described [6]. We show here that the effect ofRIF1 deletion is epistatic to loss of Tel1 or Mec1 (ATMand ATR kinases), does not affect the intra-S phasecheckpoint, and operates through a different pathwaythan the silencing protein Sir3. Deletion of a normallydormant telomere-proximal replication origin exerts asimilar effect on replication timing as does deletion ofRIF1, and these two effects are additive. Strikingly, deletionof RIF1 partially suppresses temperature-sensitivemutations in a number of essential genes that encoderegulators of DNA replication initiation, without affectingthe levels of the relevant gene products, several ofwhich are present in limiting amounts.ConclusionsThe budding yeast telomere-binding protein Rif1 is shownhere to be a global regulator of DNA replication initiationwhose loss leads to precocious replication of subtelomericdomains in the budding yeast. This appears to be a highlyconserved function of Rif1, since its homologs haverecently been shown to exert related effects in both fissionyeast and mammalian cells [7-9]. Experiments will bedescribed aimed at understanding the mechanistic basis ofthe effect of Rif1 on replication timing.