TY - JOUR
T1 - Checkpoint activation by Spd1
T2 - a competition-based system relying on tandem disordered PCNA binding motifs
AU - Olsen, Johan G
AU - Prestel, Andreas
AU - Kassem, Noah
AU - Broendum, Sebastian S
AU - Shamim, Hossain Mohammad
AU - Simonsen, Signe
AU - Grysbæk, Martin
AU - Mortensen, Josefine
AU - Rytkjær, Louise Lund
AU - Haxholm, Gitte W
AU - Marabini, Riccardo
AU - Holmberg, Christian
AU - Carr, Antony M
AU - Crehuet, Ramon
AU - Nielsen, Olaf
AU - Kragelund, Birthe B
PY - 2024
Y1 - 2024
N2 - 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.
AB - 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.
U2 - 10.1093/nar/gkae011
DO - 10.1093/nar/gkae011
M3 - Journal article
C2 - 38261971
VL - 52
SP - 2030
EP - 2044
JO - Nucleic acids symposium series
JF - Nucleic acids symposium series
SN - 0261-3166
IS - 4
ER -