Rif1 Binding and Control of Chromosome-Internal DNA Replication Origins Is Limited by Telomere Sequestration

Abstract

The Saccharomyces cerevisiae telomere-binding protein Rif1 plays an evolutionarily conserved role in control of DNA replication timing by promoting PP1-dependent dephosphorylation of replication initiation factors. However, ScRif1 binding outside of telomeres has never been detected, and it has thus been unclear whether Rif1 acts directly on the replication origins that it controls. Here, we show that, in unperturbed yeast cells, Rif1 primarily regulates late-replicating origins within 100 kb of a telomere. Using the chromatin endogenous cleavage ChEC-seq technique, we robustly detect Rif1 at late-replicating origins that we show are targets of its inhibitory action. Interestingly, abrogation of Rif1 telomere association by mutation of its Rap1-binding module increases Rif1 binding and origin inhibition elsewhere in the genome. Our results indicate that Rif1 inhibits replication initiation by interacting directly with origins and suggest that Rap1-dependent sequestration of Rif1 increases its effective concentration near telomeres, while limiting its action at chromosome-internal sites.

The Role of Rif1 in telomere length regulation is separable from its role in origin firing

“…Deletion of RIF1 activates telomeric origin firing (Dave et al, 2014;Mattarocci et al, 2014;Peace et al, 2014;Hafner et al, 2018). This origin activation requires Rif1 binding to PP1 through canonical binding motifs RVxF and SILK, (Dave et al, 2014;Hiraga et al, 2014;Mattarocci et al, 2014).…”

“…Analysis across the entire Chromosome VI showed that the left telomeric origins also showed slight increase in activation in rif1∆ and rif1-pp1bs (Supplemental Figure 1E relating to Figure 1). To determine the extent of activation of each origin, we used a method similar to (Hafner et al, 2018) and compared the relative copy number at each origin in rif1∆ and rif1-pp1bs to WT. As expected, there was higher relative copy number at most origins in the rif1∆ and rif1-pp1bs mutants, with the majority of the change seen in typically late/inefficient origins, demonstrated by a low relative copy number in WT cells ( Figure 1D).…”

Dynamic relocalization of replication origins by Fkh1 requires execution of DDK function and Cdc45 loading at origins

“…In addition to subtelomeric origins, Rif1 regulates and associates with origins distal from telomeres and with the nuclear envelope, and therefore could potentially tether origins to the periphery (Fig. 7A) (Hafner et al, 2018;Park et al, 2011;Peace et al, 2014). Rif1's interacts with Dbf4 and with the counteracting PP1 phosphatase, suggesting that the Rif1--origin interaction may be down--regulated by DDK-dependent phosphorylation of MCM proteins and/or Rif1 (Dave et al, 2014;Hiraga et al, 2014;Mattarocci et al, 2014).…”

“…In budding and fission yeast, specific mechanisms defining replication timing are linked with chromosomal domain organization (reviewed in (Aparicio, 2013;Yamazaki et al, 2013). Rif1, which is highly enriched at telomeres, is globally responsible for delayed replication timing of subtelomeric domains as well as internal late--replicating domains (Hafner et al, 2018;Hayano et al, 2012;Peace et al, 2014;Tazumi et al, 2012). Rif1 acts by directly antagonizing replication initiation triggered by Dbf4--dependent kinase (DDK) phosphorylation of MCM helicase proteins (Dave et al, 2014;Hiraga et al, 2014;Mattarocci et al, 2014).…”

The Ty1 integrase nuclear localization signal is necessary and sufficient for retrotransposon targeting to tRNA genes

“…This scattered dispersion may explain why we have failed to detect IN1 and IN5 by ChIP-seq in these regions. High-resolution mapping of DNA binding sites will be required to address this point (Hafner et al, 2018;Meers et al, 2019).…”