Abstract:Rif1, identified as a regulator of telomerase in yeast, is an evolutionarily conserved protein and functions in diverse processes including telomere length regulation, epigenetic gene regulation, anti-checkpoint activity, DNA repair and establishing timing of firing at replication origins. Previously we had identified that all Rif1 homologues have PP1 interacting SILK-RVxF motif. In the present study, we show that Drosophila Rif1 is essential for normal fly development and loss of dRif1 impairs temporal regula… Show more
“…1E). This observation, together with genetic evidence from yeast 15, 26, 27 and Drosophila 16 , suggests that Rif1 may itself be a regulatory subunit of PP1. To explore this hypothesis, we have further characterized the Rif1-PP1 interaction in molecular detail.Figure 1Rif1-associated complexes are enriched for chromatin-related proteins and PP1 substrates/regulators.…”
Rif1 is a conserved protein that plays essential roles in orchestrating DNA replication timing, controlling nuclear architecture, telomere length and DNA repair. However, the relationship between these different roles, as well as the molecular basis of Rif1 function is still unclear. The association of Rif1 with insoluble nuclear lamina has thus far hampered exhaustive characterization of the associated protein complexes. We devised a protocol that overcomes this problem, and were thus able to discover a number of novel Rif1 interactors, involved in chromatin metabolism and phosphorylation. Among them, we focus here on PP1. Data from different systems have suggested that Rif1-PP1 interaction is conserved and has important biological roles. Using mutagenesis, NMR, isothermal calorimetry and surface plasmon resonance we demonstrate that Rif1 is a high-affinity PP1 adaptor, able to out-compete the well-established PP1-inhibitor I2 in vitro. Our conclusions have important implications for understanding Rif1 diverse roles and the relationship between the biological processes controlled by Rif1.
“…1E). This observation, together with genetic evidence from yeast 15, 26, 27 and Drosophila 16 , suggests that Rif1 may itself be a regulatory subunit of PP1. To explore this hypothesis, we have further characterized the Rif1-PP1 interaction in molecular detail.Figure 1Rif1-associated complexes are enriched for chromatin-related proteins and PP1 substrates/regulators.…”
Rif1 is a conserved protein that plays essential roles in orchestrating DNA replication timing, controlling nuclear architecture, telomere length and DNA repair. However, the relationship between these different roles, as well as the molecular basis of Rif1 function is still unclear. The association of Rif1 with insoluble nuclear lamina has thus far hampered exhaustive characterization of the associated protein complexes. We devised a protocol that overcomes this problem, and were thus able to discover a number of novel Rif1 interactors, involved in chromatin metabolism and phosphorylation. Among them, we focus here on PP1. Data from different systems have suggested that Rif1-PP1 interaction is conserved and has important biological roles. Using mutagenesis, NMR, isothermal calorimetry and surface plasmon resonance we demonstrate that Rif1 is a high-affinity PP1 adaptor, able to out-compete the well-established PP1-inhibitor I2 in vitro. Our conclusions have important implications for understanding Rif1 diverse roles and the relationship between the biological processes controlled by Rif1.
“…One example where the mitotic surveillance pathway must be inactive is in early mouse embryos, which proliferate in the absence of centrosomes until the 64-cell stage [30], before becoming sensitive to centrosome loss later in development [6]. Additionally, it is clear that the mitotic surveillance pathway is not present in flies, which lack clear 53BP1 and USP28 homologues [31, 32]. Thus, while the spindle assembly checkpoint serves to guard against chromosome segregation errors and is conserved from yeast to humans, the mitotic surveillance pathway may be restricted to vertebrates.…”
Section: Concluding Remarks: a New Mode Of Mitotic Surveillancementioning
Cells have evolved certain precautions to preserve their genomic content during mitosis and avoid potentially oncogenic errors. Aside from the well-established DNA damage and spindle assembly checkpoints, recent observations have identified an additional mitotic fail safe, referred to as the mitotic surveillance pathway. This pathway triggers a cell cycle arrest to block the growth of potentially unfit daughter cells, and is activated by both prolonged mitosis and centrosome loss. Recent genome-wide screens surprisingly revealed that 53BP1 and USP28 act upstream of p53 to mediate signaling through the mitotic surveillance pathway. Here, we review advances in our understanding of this fail-safe, and discuss how 53BP1 and USP28 adopt non-canonical roles to function in this pathway.
“…Rif1 is highly conserved in eukaryotes [24, 25] and has more recently been shown to be a regulator of DNA replication initiation in yeast, flies and mammals [26–33]. We and others found that Rif1, through its conserved RVxF/SILK motifs, interacts with protein phosphatase 1 (PP1; Glc7 in budding yeast), and that this interaction is crucial for inhibition of replication origin firing by counteracting the activity of the Dbf4-dependent kinase (DDK) [26, 27, 30].…”
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.
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