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/DNAtrabectedin ternary complex, stabilized by Arg961, results in cell death. (Cancer Res 2006; 66(16): 8155-62)

Transcription is a major obstacle for replication fork (RF) progression and a cause of genome instability. Part of this instability is mediated by cotranscriptional R loops, which are believed to increase by suboptimal assembly of the nascent messenger ribonucleoprotein particle (mRNP). However, no clear evidence exists that heterogeneous nuclear RNPs (hnRNPs), the basic mRNP components, prevent R-loop stabilization. Here we show that yeast Npl3, the most abundant RNA-binding hnRNP, prevents R-loop-mediated genome instability. npl3D cells show transcription-dependent and R-loop-dependent hyperrecombination and genome-wide replication obstacles as determined by accumulation of the Rrm3 helicase. Such obstacles preferentially occur at long and highly expressed genes, to which Npl3 is preferentially bound in wild-type cells, and are reduced by RNase H1 overexpression. The resulting replication stress confers hypersensitivity to double-strand break-inducing agents. Therefore, our work demonstrates that mRNP factors are critical for genome integrity and opens the option of using them as therapeutic targets in anti-cancer treatment.

Cell wall mannoproteins are largely responsible for the adhesive properties and immunomodulation ability of the fungal pathogen Candida albicans. The outer chain extension of yeast mannoproteins occurs in the lumen of the Golgi apparatus. GDP-mannose must first be transported from the cytosol into the Golgi lumen, where mannose is transferred to mannans. GDP is hydrolyzed by a GDPase, encoded by GDA1, to GMP, which then exits the Golgi lumen in a coupled, equimolar exchange with cytosolic GDP-mannose. We isolated and disrupted the C. albicans homologue of the Saccharomyces cerevisiae GDA1 gene in order to investigate its role in protein mannosylation and pathogenesis. CaGda1p shares four apyrase conserved regions with other nucleoside diphosphatases. Membranes prepared from the C. albicans disrupted gda1/gda1 strain had a 90% decrease in the ability to hydrolyze GDP compared to wild type. The gda1/gda1 mutants showed a severe defect in O-mannosylation and reduced cell wall phosphate content. Other cell wall-related phenotypes are present, such as elevated chitin levels and increased susceptibility to attack by ␤-1,3-glucanases. Our results show that the C. albicans organism contains ␤-mannose at their nonreducing end, differing from S. cerevisiae, which has only ␣-linked mannose residues in its O-glycans. Mutants lacking both alleles of GDA1 grow at the same rate as the wild type but are partially blocked in hyphal formation in Lee solid medium and during induction in liquid by changes in temperature and pH. However, the mutants still form normal hyphae in the presence of serum and N-acetylglucosamine and do not change their adherence to HeLa cells. Taken together, our data are in agreement with the hypothesis that several pathways regulate the yeast-hypha transition. Gda1/gda1 cells offer a model for discriminating among them.