Checkpoint activation by Spd1: a competition-based system relying on tandem disordered PCNA binding motifs

Johan G Olsen, Andreas Prestel, Noah Kassem, Sebastian S Broendum, Hossain Mohammad Shamim, Signe Simonsen, Martin Grysbæk, Josefine Mortensen, Louise Lund Rytkjær, Gitte W Haxholm, Riccardo Marabini, Christian Holmberg, Antony M Carr, Ramon Crehuet, Olaf Nielsen, Birthe B Kragelund

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Abstract

DNA regulation, replication and repair are processes fundamental to all known organisms and the sliding clamp proliferating cell nuclear antigen (PCNA) is central to all these processes. S-phase delaying protein 1 (Spd1) from S. pombe, an intrinsically disordered protein that causes checkpoint activation by inhibiting the enzyme ribonucleotide reductase, has one of the most divergent PCNA binding motifs known. Using NMR spectroscopy, in vivo assays, X-ray crystallography, calorimetry, and Monte Carlo simulations, an additional PCNA binding motif in Spd1, a PIP-box, is revealed. The two tandemly positioned, low affinity sites exchange rapidly on PCNA exploiting the same binding sites. Increasing or decreasing the binding affinity between Spd1 and PCNA through mutations of either motif compromised the ability of Spd1 to cause checkpoint activation in yeast. These results pinpoint a role for PCNA in Spd1-mediated checkpoint activation and suggest that its tandemly positioned short linear motifs create a neatly balanced competition-based system, involving PCNA, Spd1 and the small ribonucleotide reductase subunit, Suc22R2. Similar mechanisms may be relevant in other PCNA binding ligands where divergent binding motifs so far have gone under the PIP-box radar.
OriginalsprogEngelsk
BogserieNucleic acids symposium series
Vol/bind52
Udgave nummer4
Sider (fra-til)2030–2044
Antal sider15
ISSN0261-3166
DOI
StatusUdgivet - 2024

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