Abstract
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.
Highlights
IntroductionRif[1] phosphatase 1 (PP1) docking motifs are localized at the N-terminus of the protein
Throughout evolution, Rif[1] has maintained one or more motifs that potentially mediate the interaction with the Ser/Thr protein phosphatase 1 (PP1), such as the PP1 docking motif [K/R][V/I]xF and the conserved SILK sequence, an additional binding motif necessary for a subset of PP1-interacting proteins[19, 24, 25]
The characterization of endogenous Rif1-associated complexes has been hampered by the lack of optimized antibodies and the subnuclear localization of this protein, which render it extremely challenging to isolate
Summary
Rif[1] PP1 docking motifs are localized at the N-terminus of the protein Their function has been validated through mutagenesis, demonstrating that Rif[1] counteracts DDK activity on the replicative MCM4 helicase through its interaction with PP115, 26, 27. Human and mouse Rif1s contain at least two different potential PP1-interacting peptides, canonical RVSF/SILK motifs in CRI and an additional KIAF motif in the HEAT repeats. These structural features indicate that the interaction of Rif[1] with PP1 may represent a central aspect of Rif[1] biological role throughout evolution. Our data classify Rif[1] as a bona fide PP1 regulatory subunit
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