Trabectedin (Yondelis) is a potent antitumor drug that has the unique characteristic of killing cells by poisoning the DNA nucleotide excision repair (NER) machinery. The basis for the NER-dependent toxicity has not yet been elucidated but it has been proposed as the major determinant for the drug's cytotoxicity. To study the in vivo mode of action of trabectedin and to explore the role of NER in its cytotoxicity, we used the fission yeast Schizosaccharomyces pombe as a model system. Treatment of S. pombe wild-type cells with trabectedin led to cell cycle delay and activation of the DNA damage checkpoint, indicating that the drug causes DNA damage in vivo. DNA damage induced by the drug is mostly caused by the NER protein, Rad13 (the fission yeast orthologue to human XPG), and is mainly repaired by homologous recombination. By constructing different rad13 mutants, we show that the DNA damage induced by trabectedin depends on a 46–amino acid region of Rad13 that is homologous to a DNA-binding region of human nuclease FEN-1. More specifically, an arginine residue in Rad13 (Arg961), conserved in FEN1 (Arg314), was found to be crucial for the drug's cytotoxicity. These results lead us to propose a model for the action of trabectedin in eukaryotic cells in which the formation of a Rad13/DNA-trabectedin ternary complex, stabilized by Arg961, results in cell death. (Cancer Res 2006; 66(16): 8155-62)

In fission yeast, the cyclin‐dependent kinase (CDK) inhibitor p25rum1 is a key regulator of progression through the G1 phase of the cell cycle. We show here that p25rum1 protein levels are sharply periodic. p25rum1 begins to accumulate at anaphase, persists in G1 and is destroyed during S phase. p25rum1 is stabilized and polyubiquitinated in a mutant defective in the 26S proteasome, suggesting that its degradation normally occurs through the ubiquitin‐dependent 26S proteasome pathway. Phosphorylation of p25rum1 by cdc2–cyclin complexes at residues T58 and T62 is important to target the protein for degradation. Mutation of one or both of these residues to alanine causes stabilization of p25rum1 and induces a cell cycle delay in G1 and polyploidization due to occasional re‐initiation of DNA replication before mitosis. The CDK–cyclin complex cdc2–cig1, which is insensitive to p25rum1 inhibition, seems to be the main kinase that phosphorylates p25rum1. Phosphorylation of p25rum1 in S phase and G2 serves as the trigger for p25rum1 proteolysis. Thus, periodic accumulation and degradation of the CDK inhibitor p25rum1 in G1 plays a role in setting a threshold of cyclin levels important in determining the length of the pre‐Start G1 phase and in ensuring the correct order of cell cycle events.

Eukaryotic cells coordinate cell size with cell division by regulating the length of the G1 and G2 phases of the cell cycle. In fission yeast, the length of the G1 phase depends on a precise balance between levels of positive (cig1, cig2, puc1, and cdc13 cyclins) and negative (rum1 and ste9-APC) regulators of cdc2. Early in G1, cyclin proteolysis and rum1 inhibition keep the cdc2/cyclin complexes inactive. At the end of G1, the balance is reversed and cdc2/cyclin activity down-regulates both rum1 and the cyclin-degrading activity of the APC. Here we present data showing that the puc1 cyclin, a close relative of the Cln cyclins in budding yeast, plays an important role in regulating the length of G1. Fission yeast cells lacking cig1 and cig2 have a cell cycle distribution similar to that of wild-type cells, with a short G1 and a long G2. However, when the puc1(+) gene is deleted in this genetic background, the length of G1 is extended and these cells undergo S phase with a greater cell size than wild-type cells. This G1 delay is completely abolished in cells lacking rum1. Cdc2/puc1 function may be important to down-regulate the rum1 Cdk inhibitor at the end of G1.