Irina Marinova:
Suppressor Analysis of CRL4Cdt2 Defective and cdc48-353 Temperature Sensitive Mutants in Fission Yeast

Date: 24-08-2016    Supervisor: Olaf Nielsen & Rasmus Hartmann-Petersen

Part 1
CRL4Cdt2 E3 ligase is a key regulator of cellular proliferation and genome integrity, as it promotes the degradation of proteins involved in cell cycle progression, DNA replication and repair. In fission yeast the small intrinsically disordered protein Spd1 is targeted for degradation upon entry into S-phase and following DNA damage via CRL4Cdt2-mediated ubiquitylation, which also requires interaction with proliferating cell nuclear antigen (PCNA). Spd1 is a negative regulator of ribonucleotide reductase (RNR), the activity of which is required for deoxyribonucleotide (dNTP) synthesis. CRL4Cdt2 mutants, which fail to degrade Spd1, have decreased dNTP pool and their viability is dependent on constitutive activation of the DNA integrity checkpoint. However, accumulation of Spd1 in RNR mutants with elevated dNTP levels still causes checkpoint activation and dependency. Here, we show that Spd1 amino acid residues V40 and S43 are important for its function as an inhibitor of DNA synthesis, as the spd1-V40G and spd1-S43L mutants were identified as spontaneous suppressors of the defective phenotypes exhibited by cells with abrogated CRL4Cdt2 pathway. We confirm that these mutations alleviate the checkpoint dependency, the DNA damage sensitivity and the meiotic defects associated with Spd1 accumulation. Further analysis showed that whereas the V40G and S43L substitutions do not have a significant impact on Suc22R2 nuclear import function of Spd1, they affect the interaction between Spd1 and PCNA. Our results provide evidence that excess Spd1 causes replication stress and genome instability by inhibiting PCNA. Furthermore, we examined the potential regulation of Spd1 function by Gad8-mediated phosphorylation of residue S43.

Part 2
Cdc48/p97 is a ring-shaped homohexameric chaperone-like complex involved in numerous cellular processes, including protein degradation, cell cycle control, DNA repair, and vesicle fusion. The cdc48 gene is essential in fission yeast and mutations or changes in Cdc48/p97 protein expression have been linked to neurological disorders and cancer in humans. To gain further insight into the function of Cdc48/p97, we performed a screen for pseudo revertants of the cdc48-353 temperature-sensitive fission yeast mutant. Altogether, we isolated 28 independent spontaneous cdc48-353 suppressors that had also acquired a cold-sensitive phenotype. Using whole genome sequencing approach, we showed that these suppressors were all second site intragenic cdc48 mutants. Mapping the suppressor mutations on the Cdc48 structure revealed that whereas the original G338D lesion was located near the central pore of the hexameric ring, the suppressor mutations were positioned at the subunit-subunit and inter-domain boundaries. Furthermore, we isolated a suppressor which had not acquired a cold-sensitive phenotype and carried an extragenic frame shift mutation in the ufd1 gene, which encodes a known co-factor of Cdc48. Collectively, our results provide evidence that the structural stability of the Cdc48-353 hexamer is perturbed at the restrictive temperature, but stabilized in the suppressors.