Riccardo Marabini:
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Ribonucleotide reductase (RNR) and proliferating cell nuclear antigen (PCNA) are two essential proteins involved in DNA replication. RNR catalyzes the last and rate limiting step of the deoxyribonucleotide biosynthetic pathway. The dysregulation of RNR has been related to higher mutation rate and the onset of cancer. PCNA is part of the DNA sliding clamp family, and it is a processivity factor that enhances the activity of DNA polymerases both in DNA replication and repair. In the last two decades, a family of five small intrinsically disordered proteins (IDP) has been identified and genetically characterized in budding and fission yeast. Within this protein family Dif1 (from S. cerevisiae) and Spd1 (from S. pombe) were analyzed in this study. These proteins were previously found to interact with and regulate the activity of RNR and Spd1 was also linked to PCNA dependent signaling for degradation. Although a fundamental functional genetic characterization of these IDP is available in the literature, structural and residue-specific information was poorly described. Furthermore, a careful correlation between the known functions of the RNR inhibitors and eventual short linear motifs (SLiMs) was almost completely absent. SLiMs are compact monopartite interaction modules formed by linear short stretches of residues. In this work we combined structural and SLiMs studies, genetics analysis, and post-translational modification assays to characterize the IDP, RNR and PCNA network. First, we showed by NMR and genetic studies that Dif1 strongly inhibits DNA replication in S. pombe and interacts in vitro and in vivo with RNR and PCNA. In these interactions, two overlapping short linear motifs were involved. In addition, we revealed that in vitro mono-phosphorylation of Dif1 induce a slight transient helix stabilization. Moreover, we provided experimental evidence indicating that specific Spd1 phosphorylation might work as a regulatory switch that controls three SLiMs involved in the binding to RNR and PCNA. Last but not least, we inspected more than 70 PCNA-interacting proteins motifs (PIP-motifs) revealing that disorder and high pI dominate the local context of the PIP-motifs. Concluding, a fundamental improvement in the knowledge of the regulatory network of IDP, RNR and PCNA has been obtained, and a new view of subclassification of the PIP-motifs provided.