Rif1 maintains telomeres and mediates DNA repair by encasing DNA ends

In yeast, Rif1 is part of the telosome, where it inhibits telomerase and checkpoint signaling at chromosome ends. In mammalian cells, Rif1 is not telomeric, but it suppresses DNA end resection at chromosomal breaks, promoting repair by nonhomologous end joining (NHEJ). Here, we describe crystal structures for the uncharacterized and conserved ∼125-kDa N-terminal domain of Rif1 from Saccharomyces cerevisiae (Rif1-NTD), revealing an α-helical fold shaped like a shepherd's crook. We identify a high-affinity DNA-binding site in the Rif1-NTD that fully encases DNA as a head-to-tail dimer. Engagement of the Rif1-NTD with telomeres proved essential for checkpoint control and telomere length regulation. Unexpectedly, Rif1-NTD also promoted NHEJ at DNA breaks in yeast, revealing a conserved role of Rif1 in DNA repair. We propose that tight associations between the Rif1-NTD and DNA gate access of processing factors to DNA ends, enabling Rif1 to mediate diverse telomere maintenance and DNA repair functions

On start and pause of replication fork

In my PhD thesis I describe the work I've done with my colleagues in Professor David Shore's Lab that led to the next 3 main conclusions. (1) Conserved protein Rif1 is a negative regulator of DNA replication initiation in budding yeast. Rif1 counteracts DDK-dependent activatory phosphorylation at Mcm4 and Sld3 replication initiation proteins by recruiting protein phosphatase PP1 (ScGlc7) through its RVxF/SILK motifs. (2) Replication-restraining activity of Rif1 is important for the maintenance of genome integrity. Cells lacking Rif1 have elevated origin activation in early S phase at ribosomal RNA gene tandem repeat array (rDNA), which leads to destabilization of the array and dependency on DSB repair/fork maintenance factors MRX and Mms22-Ctf4 for survival. (3) Replisome pausing factors Tof1-Csm3 promote pausing at RFB independently of replisome accessory helicase Rrm3. Replication fork pausing at proteinaceous barriers (RFBs) depends on DNA lagging strand synthesis machinery and topoisomerases I and II

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

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

Rif1 Controls DNA Replication Timing in Yeast through the PP1 Phosphatase Glc7

The Rif1 protein, originally identified as a telomere-binding factor in yeast, has recently been implicated in DNA replication control from yeast to metazoans. Here, we show that budding yeast Rif1 protein inhibits activation of prereplication complexes (pre-RCs). This inhibitory function requires two N-terminal motifs, RVxF and SILK, associated with recruitment of PP1 phosphatase (Glc7). In G1 phase, we show both that Glc7 interacts with Rif1 in an RVxF/SILK-dependent manner and that two proteins implicated in pre-RC activation, Mcm4 and Sld3, display increased Dbf4-dependent kinase (DDK) phosphorylation in rif1 mutants. Rif1 also interacts with Dbf4 in yeast two-hybrid assays, further implicating this protein in direct modulation of pre-RC activation through the DDK. Finally, we demonstrate Rif1 RVxF/SILK motif-dependent recruitment of Glc7 to telomeres and earlier replication of these regions in cells where the motifs are mutated. Our data thus link Rif1 to negative regulation of replication origin firing through recruitment of the Glc7 phosphatase

Budding Yeast Rif1 Controls Genome Integrity by Inhibiting rDNA Replication

<div><p>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 <i>rif1Δ sir2Δ</i> 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 <i>sir2Δ</i>. We find, in addition, that the viability of <i>rif1Δ</i> 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 <i>rif1Δ</i> 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.</p></div

Rif1 acts together with Sir2 to inhibit rDNA origin firing, but independently to block late origin firing.

<p>(A-B) Relative BrdU incorporation at the indicated loci (A) and 2D agarose gel electrophoresis (B) in WT, <i>rif1Δ</i>, <i>sir2Δ</i>, and <i>rif1Δ sir2Δ</i> mutant cultures released from nocodazole (G2/M) arrest into 0.2 M HU for 2 hrs. Data are presented as mean +/- SEM of three independent experiments and a t-test was used to compare the means of WT and mutant cultures. (*) P < 0.05.</p

Rif1 functions in budding yeast beyond the rDNA locus.

<p>(A) A proposed model for Rif1 effects on both DNA replication timing and DNA replication checkpoint activation in budding yeast. (B-E) Western blots of total Rad53 following HU (0.2 M for 2 hrs) or mock treatment of asynchronous cultures of the indicated mutant combinations: (B) <i>rif1-RBM</i> and <i>rif1-RVxF/SILK</i>, (C) <i>rif1Δ</i> and <i>fob1Δ</i>, (D) <i>rif1Δ</i> and <i>fob1Δ</i> in strains harboring 20 or 2 (<i>rdn1Δ</i>) rDNA repeats on chromosome XII, or (E) <i>rif1Δ</i> and <i>sir2Δ</i>. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006414#pgen.1006414.s007" target="_blank">S7 Fig</a>.</p

Schematic representation of a model for Rif1 and Sir2 function at the rDNA locus.

<p>In WT cells both Rif1-Glc7 and Sir2 inhibit DNA replication initiation at the rDNA locus thereby maintaining its integrity (A). Inactivation of Rif1-Glc7 abolishes the inhibitory dephosphorylation activity and leads to an increase in the DDK-dependent DNA replication initiation at rDNA origins and late origins throughout the genome (B). Deletion of the Sir2 histone deacetylase increases accessibility of the rDNA locus to the limited pool of DNA replication initiation factors (C). Both conditions (B and C) are accompanied by accumulation of replication forks at the Fob1-dependent RFB, leading to rDNA array instability and increased sickness in strain backgrounds deficient in fork maintenance or DNA repair.</p

rDNA instability is the major cause of synthetic lethality in <i>rif1Δ</i> cells.

<p>Overnight cultures of the indicated genotypes were serially diluted 1:10 and spotted onto solid YPAD medium or YPAD medium supplemented with phleomycin (PHL). Plates were incubated at the indicated temperature for 2–4 days before being photographed. (A) The synthetic sickness of <i>rif1Δ mre11Δ</i> cells is rescued by deletion of the <i>FOB1</i> gene. (B) The <i>rif1 RVxF/SILK</i> mutant is synthetic sick with <i>mre11Δ</i> both in normal conditions and in the presence of PHL. (C) Effect of the <i>cdc7-4</i> allele on synthetic sickness of <i>rif1Δ mre11Δ</i> mutants. (D) Effect of <i>rdn1Δ</i> on synthetic interactions of <i>mre11Δ</i> and <i>rif1Δ</i>. (E) Tetrad dissection showing the synthetic sickness of <i>sir2Δ mre11Δ</i> and its rescue by <i>fob1Δ</i>. (F) Synthetic sickness of <i>rif1Δ</i> with <i>ctf4Δ</i> and <i>mms22Δ</i> is rescued by <i>fob1Δ</i>. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006414#pgen.1006414.s005" target="_blank">S5</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006414#pgen.1006414.s006" target="_blank">S6</a> Figs.</p