Cell Cycle and Genome Stability – Department of Biology - University of Copenhagen

Cell Cycle and Genome Stability

At the restriction point (or START) in late G1, a cell can chose between alternative developmental trajectories. If conditions favour growth, the cell will enter S-phase followed by mitotic division, producing two identical daughter cells. However, under different environmental cues the cell may enter a quiescent G0 state, or undergo differentiation to produce another cell type. Our research aims at understanding key events at the restriction point. In particular, we are interested in the regulatory consequences of commitment to DNA replication in the mitotic cell cycle: how do mechanisms ensuring genome stability become activated at this point, and also how do alternative developmental pathways become blocked? For most of our studies we use the model organism fission yeast as experimental system, but we also seek to confirm the general significance of our findings by performing experiments with mammalian cells.

 

Genome stability and DNA building blocks

When cells commit to S-phase, they activate the Cul4/Ddb1 ubiquitin ligase, which regulates the turnover of key protein substrates that need to be degraded for S-phase to proceed correctly. One important substrate for this pathway is the small, unstructured protein Spd1. Spd1 is an inhibitor of the enzyme RNR (= ribonucleotide reductase), which catalyses the synthesis of dNTP DNA building blocks. Failure to degrade Spd1 in Ddb1-deficient cells, causes reduced dNTP pools, resulting in slow S-phase progression, sensitivity to DNA damaging agents and a more than 20 fold increase in spontaneous mutation rates. In addition, survival of unperturbed cells becomes reliant on intact DNA structure checkpoints.

Genome stability and heterochromatin

Mutants in Cul4 (pcu4) exhibit a more severe genome stability defect than ddb1 mutants, suggesting that Cul4 participates in ubiquitination reactions that do not require Ddb1. The Rik1 protein is homologous to Ddb1 over its entire length, and both rik1and pcu4 cells are defective in RNAi mediated formation of heterochromatin. Since heterochromatin formation in centromeric regions is important for proper chromosome segregation during mitosis, this can formally explain the observed genome stability defects. Taken together, these observations suggest that a second Cul4 based complex, containing Rik1 instead of Ddb1 ubiquinates substrates important for RNAi-mediated heterochromatin formation. Such substrates have not yet been identified.

Control of sexual differentiation

Fission yeast cells undergo sexual differentiation in response to nitrogen deprivation. In this process, haploid P- and M-cells first conjugate to form zygotes, which subsequently undergo meiosis and sporulation. Like other differentiation processes, conjugation is only possible from the pre-START window of the cell cycle. We study the mechanisms by which commitment to the mitotic cell cycle prevents differentiation. Our experiments have shown that Ste11, the key transcription factor controlling the differentiation process, becomes phosphorylated by the increase in cyclin-dependant kinase (CDK) activity that also triggers S-phase. This phosphorylation prevents Ste11 from binding to its target promoters and hence abrogates induction of differentiation-specific genes.