Budding Yeast Rif1 Controls Genome Integrity by Inhibiting rDNA Replication

Abstract

The Rif1 protein is a negative regulator of DNA replication initiation in eukaryotes. Here we show that budding yeast Rif1 inhibits DNA replication initiation at the rDNA locus. Absence of Rif1, or disruption of its interaction with PP1/Glc7 phosphatase, leads to more intensive rDNA replication. The effect of Rif1-Glc7 on rDNA replication is similar to that of the Sir2 deacetylase, and the two would appear to act in the same pathway, since the rif1Δ sir2Δ double mutant shows no further increase in rDNA replication. Loss of Rif1-Glc7 activity is also accompanied by an increase in rDNA repeat instability that again is not additive with the effect of sir2Δ. We find, in addition, that the viability of rif1Δ cells is severely compromised in combination with disruption of the MRX or Ctf4-Mms22 complexes, both of which are implicated in stabilization of stalled replication forks. Significantly, we show that removal of the rDNA replication fork barrier (RFB) protein Fob1, alleviation of replisome pausing by deletion of the Tof1/Csm3 complex, or a large deletion of the rDNA repeat array all rescue this synthetic growth defect of rif1Δ cells lacking in addition either MRX or Ctf4-Mms22 activity. These data suggest that the repression of origin activation by Rif1-Glc7 is important to avoid the deleterious accumulation of stalled replication forks at the rDNA RFB, which become lethal when fork stability is compromised. Finally, we show that Rif1-Glc7, unlike Sir2, has an important effect on origin firing outside of the rDNA locus that serves to prevent activation of the DNA replication checkpoint. Our results thus provide insights into a mechanism of replication control within a large repetitive chromosomal domain and its importance for the maintenance of genome stability. These findings may have important implications for metazoans, where large blocks of repetitive sequences are much more common.

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

“…We found that rif1D caused significantly earlier recruitment at all three of these late-replicating telomere-proximal sites ( Figure 1A, right panel) and obtained similar results for origins close to the end of chromosome I-L ( Figure S1B). This effect is not due to telomere elongation caused by chronic absence of Rif1 (as in rif1D cells), because we observed the same phenotype after only 1 hr of anchor-away depletion of Rif1 (Haruki et al, 2008;Shyian et al, 2016), during which time telomere elongation is negligible ( Figure S1C).…”

“…Conversely, for many origins farther from telomeres, and not Rap1-associated, the Rif1-RBM mutant showed higher ChEC signal compared to WT ( Figure 4B, for examples of two telomere-distal late ARSs; Figure 4C, red signal in the RBM/WT heatmap). One notable example of this effect is seen at the repetitive rDNA locus, where Rif1 inhibits the firing of an origin present in each of the 150-200 rDNA copies (Shyian et al, 2016). Two sites of enhanced Rif1-MNase cleavage are detected in the rDNA: one at the promoter of TAR1, which contains a Rap1 binding site, and a second at the rDNA origin, where no nearby Rap1 binding is observed ( Figure S4F).…”

“…Until now, all DNA replication timing experiments in rif1D strains have been performed in cells released from either a physiological (a factor pheromone) ( Figure 1) or a genetic (cdc7) cell-cycle block (Davé et al, 2014;Hiraga et al, 2014;Lian et al, 2011;Mattarocci et al, 2014;Peace et al, 2014;Shyian et al, 2016). Such blocks might affect the replication initiation regulatory machinery, which is coupled to the cell cycle.…”

Cell volume homeostatically controls the rDNA repeat copy number and rRNA synthesis rate in yeast

“…We have also found that fresh cln3 mutants with a small rDNA copy number have no growth defects (not shown). The defects in rDNA silencing by Sir2 affect DNA replication and promote increased ARS firing and DNA recombination, which lead to genome instability and lethality [46]. The decreased Sir2 activity in the cln3 low repeat copy number may cause increased cell death.…”

Promotion of hyperthermic-induced rDNA hypercondensation inSaccharomyces cerevisiae

“…Due to this highly repetitive structure and active transcriptional status, rDNA is the most recombinogenic, and therefore mutagenic, site within the eukaryotic genome (Nomura, 2001; Tomson et al, 2006; Pal et al, 2018; Kwan et al, 2016). The importance of maintaining rDNA locus stability is highlighted by the fact that DNA replication forks are programmed to stall within rDNA, precluding catastrophic head-on collision of replication and transcription complexes (Biswas et al, 2017; Weitao et al, 2003; Shyian et al, 2016). Furthermore, rDNA transcription rates, and even nucleolar size, are intimately coupled to changes in nutrient levels, revealing that rDNA plays a central role in responding to environmental cues (Li et al, 2006; Tsang et al, 2007; Wang et al, 2016).…”