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 -