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

Background

DNA replication in eukaryotes is initiated from specific chromosomal sites (origins) that fire in a defined, cell type-specific temporal pattern. This replication program appears to be under epigenetic control through mechanisms that are still poorly understood.

Materials and methods

Studies were done in Saccharomyces cerevisiae (W303 background) using standard genetic and molecular methods. Chromatin immunoprecipitation assays were performed in strains in which genes encoding DNA polymerase 1 or 2 were epitope-tagged at their endogenous loci.

Results

We showed previously that telomere TG-repeat tract length exerts an epigenetic effect in cis on the activity of nearby subtelomeric replication origins, such that a shortened telomere will replicate earlier [1]. Here we show that deletion of the RIF1 gene, which encodes a telomere-specific Rap1-interacting protein involved in telomere length regulation and telomere “capping” [2–5], also leads to premature replication of two different subtelomeric regions examined. A similar effect of RIF1 deletion on other subtelomeric regions has recently been described [6]. We show here that the effect of RIF1 deletion is epistatic to loss of Tel1 or Mec1 (ATM and ATR kinases), does not affect the intra-S phase checkpoint, and operates through a different pathway than the silencing protein Sir3. Deletion of a normally dormant telomere-proximal replication origin exerts a similar effect on replication timing as does deletion of RIF1, and these two effects are additive. Strikingly, deletion of RIF1 partially suppresses temperature-sensitive mutations in a number of essential genes that encode regulators of DNA replication initiation, without affecting the levels of the relevant gene products, several of which are present in limiting amounts.

Conclusions

The budding yeast telomere-binding protein Rif1 is shown here to be a global regulator of DNA replication initiation whose loss leads to precocious replication of subtelomeric domains in the budding yeast. This appears to be a highly conserved function of Rif1, since its homologs have recently been shown to exert related effects in both fission yeast and mammalian cells [7–9]. Experiments will be described aimed at understanding the mechanistic basis of the effect of Rif1 on replication timing.