Rif1 controls DNA replication by directing Protein Phosphatase 1 to reverse Cdc7-mediated phosphorylation of the MCM complex

Abstract

Initiation of eukaryotic DNA replication requires phosphorylation of the MCM complex by Dbf4-dependent kinase (DDK), composed of Cdc7 kinase and its activator, Dbf4. We report here that budding yeast Rif1 (Rap1-interacting factor 1) controls DNA replication genome-wide and describe how Rif1 opposes DDK function by directing Protein Phosphatase 1 (PP1)-mediated dephosphorylation of the MCM complex. Deleting RIF1 partially compensates for the limited DDK activity in a cdc7-1 mutant strain by allowing increased, premature phosphorylation of Mcm4. PP1 interaction motifs within the Rif1 N-terminal domain are critical for its repressive effect on replication. We confirm that Rif1 interacts with PP1 and that PP1 prevents premature Mcm4 phosphorylation. Remarkably, our results suggest that replication repression by Rif1 is itself also DDK-regulated through phosphorylation near the PP1-interacting motifs. Based on our findings, we propose that Rif1 is a novel PP1 substrate targeting subunit that counteracts DDK-mediated phosphorylation during replication. Fission yeast and mammalian Rif1 proteins have also been implicated in regulating DNA replication. Since PP1 interaction sites are evolutionarily conserved within the Rif1 sequence, it is likely that replication control by Rif1 through PP1 is a conserved mechanism.

Replication timing is regulated by the number of MCMs loaded at origins

“…However, the fact that we observe large numbers of MCMs loaded at late-replicating telomeres (Fig. 1A) suggests that heterochromatin can delay the firing of loaded MCMs, perhaps by counteracting the activity of rate-limiting activators (Hiraga et al 2014;Mattarocci et al 2014).…”

“…In budding yeast, proximity to telomeric heterochromatin is both necessary and sufficient to delay origin firing (Ferguson and Fangman 1992). This effect is regulated by Rif1-dependent recruitment of protein phosphatase I, which may antagonize origin activation by the cyclin-and DBF4-dependent kinases (Lian et al 2011;Davé et al 2014;Hiraga et al 2014;Mattarocci et al 2014). Chromatin structure-in particular, histone deacetylation by Sir2 and Rpd3-also affects the timing of nontelomeric origins (Knott et al 2009(Knott et al , 2012Peace et al 2014;Yoshida et al 2014).…”

The selective inhibition of protein phosphatase-1 results in mitotic catastrophe and impaired tumor growth

“…PP1 forms heterodimeric or heterotrimeric complexes with >200 PP1-interacting proteins (PIPs), which function as substrate-targeting or -specifying subunits and activity regulators. PP1 has established substrates in all phases of the cell cycle, including the retinoblastoma protein in the G1 phase (Ludlow et al, 1993), the helicase MCM4 in S-phase (Hiraga et al, 2014), protein phosphatase Cdc25 in G2 phase (Margolis et al, 2006) and protein kinases Aurora A and Aurora B in M-phase (Bollen et al, 2009).…”

Unexpected roles of a shugoshin protein at subtelomeres

“…The other possibility is that Sgo2 acts at the subtelomeres independently of the knobs. As described in Masai et al's review, the telomere-binding protein Rif1 regulates the replication timing of late origins by interacting with PP1 phosphatase to counteract the action of Dbf4-dependent kinase (DDK) at the initiation step of replication (Davé et al, 2014;Hiraga et al, 2014;Mattarocci et al, 2014). Interestingly, Sgo1 forms a complex with PP2A phosphatase to protect cohesins in S. pombe, S. cerevisiae and mammals (Kitajima et al, 2006;Riedel et al, 2006;Tang et al, 2006;, and human Sgo2 also interacts with PP2A for the protection of cohesion (Tanno et al, 2010).…”