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

Area of Science:

• Molecular Biology
• Cell Cycle Regulation
• Protein-Protein Interactions

Background:

• Proliferating cell nuclear antigen (PCNA) is essential for DNA regulation, replication, and repair.
• S-phase delaying protein 1 (Spd1) from S. pombe is an intrinsically disordered protein that activates checkpoints by inhibiting ribonucleotide reductase.
• Spd1 possesses a highly divergent PCNA binding motif, previously not fully characterized.

Purpose of the Study:

• To investigate the interaction between Spd1 and PCNA, focusing on Spd1's unique binding motifs.
• To elucidate the role of PCNA in Spd1-mediated checkpoint activation.
• To understand the mechanism of Spd1's function in DNA replication and cell cycle control.

Main Methods:

• Nuclear Magnetic Resonance (NMR) spectroscopy
• In vivo assays in yeast
• X-ray crystallography
• Calorimetry
• Monte Carlo simulations

Main Results:

• An additional PCNA binding motif, a PIP-box, was identified in Spd1, positioned tandemly with the known motif.
• These two low-affinity binding sites on Spd1 rapidly exchange on PCNA, utilizing the same binding pockets.
• Mutations affecting the binding affinity of either motif impaired Spd1's checkpoint activation function in yeast.

Conclusions:

• PCNA plays a critical role in Spd1-mediated checkpoint activation.
• Spd1 utilizes tandemly arranged short linear motifs, creating a balanced competition system involving PCNA, Spd1, and the small ribonucleotide reductase subunit (Suc22R2).
• This mechanism may be relevant for other PCNA-binding proteins with divergent motifs that have been overlooked.