Novel DNA repair pathways required for cell survival following exposure to topoisomerase II targeting agents.

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Agents targeting DNA topoisomerases are active against a wide range of human tumors. Stabilization of covalent complexes, converting topoisomerases into DNA damage, is an essential aspect of cell killing by these drugs. We have taken advantage of newly developed yeast genomic tools to identify genes encoding DNA repair or DNA damage tolerance functions that play key roles in sensitivity to drugs targeting topoisomerases. A consortium of yeast investigators has constructed S. cerevisiae strains that carry deletions of every open reading frame of the yeast genome. Deletions of all genes that are not essential for viability have been screened for a wide range of phenotypes, including sensitivity to a variety of different DNA damaging agents. We are using this deletion set to identify all yeast genes that confer sensitivity to drugs targeting DNA topoisomerase II. Unfortunately, most drugs that target DNA topoisomerase II do not accumulate in wild type yeast cells to high enough. levels to affect cell growth. We have found that vectors that overexpress topoisomerase II, or vectors that express a topoisomerase II mutant that is hypersensitive to DNA intercalating agents confers a high enough level of sensitivity to mAMSA to allow efficient screening of the yeast deletion strains. As a first step, we have examined the mAMSA sensitivity of a set of strains deleted for genes that confer hypersensitivity to ionizing radiation. We found that several genes also confer high levels of sensitivity to mAMSA including strains with mutations in HPR5, a helicase involved in funneling DNA lesion into recombination-repair pathways, ASF1, a chromatin assembly factor that is required for gene silencing, and CTF4, a DNA polymerase alpha binding protein important for genome stability. Deletions of these genes were also introduced into yeast strains with enhanced drug accumulation. All three mutants described above conferred hypersensitivity to mAMSA and etoposide. Interestingly, all three mutants also showed only modest effects on sensitivity to the topoisomerase I targeting agent camptothecin. These results indicate the utility of genomic approaches using model organisms to identifying pathways for sensitivity to chemotherapeutic agents. Our results may also provide important clues to the differences in cellular responses to drugs targeting such as camptothecin, that target topoisomerase I and drugs such as etoposide that target topoisomerase II. (Supported by the American Lebanese Syrian Assoicated Charities (ALSAC) and NIH grant CA 82313.)