Roles of nonhomologous end-joining pathways in surviving topoisomerase II-mediated DNA damage.

Topoisomerase II is a target for clinically active anticancer drugs. Drugs targeting these enzymes act by preventing the religation of enzyme-DNA covalent complexes leading to protein-DNA adducts that include single- and double-strand breaks. In mammalian cells, nonhomologous repair pathways are critical for repairing topoisomerase II-mediated DNA damage. Because topoisomerase II-targeting agents, such as etoposide, can also induce chromosomal translocations that can lead to secondary malignancies, understanding nonhomologous repair of topoisomerase II-mediated DNA damage may help to define strategies that limit this critical side effect on an important class of anticancer agents. Using Saccharomyces cerevisiae as a model eukaryote, we have determined the contribution of genes required for nonhomologous end-joining (NHEJ) for repairing DNA damage arising from treatment with topoisomerase II poisons, such as etoposide and 4'-(9-acridinylamino)methanesulfon-m-anisidide (mAMSA). To increase cellular sensitivity to topoisomerase II poisons, we overexpressed either wild-type or drug-hypersensitive alleles of yeast topoisomerase II. Using this approach, we found that yku70 (hdf1), yku80 (hdf2), and other genes required for NHEJ were important for cell survival following exposure to etoposide. The clearest increase in sensitivity was observed with cells overexpressing an etoposide-hypersensitive allele of TOP2 (Ser740Trp). Hypersensitivity was also seen in some end-joining defective mutants exposed to the intercalating agent mAMSA, although the increase in sensitivity was less pronounced. To confirm that the increase in sensitivity was not solely due to the elevated expression of TOP2 or due to specific effects of the drug-hypersensitive TOP2 alleles, we also found that deletion of genes required for NHEJ increased the sensitivity of rad52 deletions to both etoposide and mAMSA. Taken together, these results show a clear role for NHEJ in the repair of DNA damage induced by topoisomerase II-targeting agents and suggest that this pathway may participate in translocations generated by drugs, such as etoposide.