Roles of nonhomologous end-joining pathways in surviving topoisomerase II–mediated DNA damage Mobeen Malik, Karin C. Nitiss, Vanessa Enriquez-Rios, and John L. Nitiss Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, Memphis, Tennessee Abstract 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 Saccharomy- ces 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 etopo- side and 4¶-(9-acridinylamino)methanesulfon-m-anisidide (mAMSA). To increase cellular sensitivity to topoisomer- ase 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 over- expressing an etoposide-hypersensitive allele of TOP2 (Ser 740 Trp). 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-hypersen- sitive 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. [Mol Cancer Ther 2006;5(6):1405 – 14] Introduction DNA topoisomerases are essential enzymes required to maintain normal DNA topology in cells (reviewed in refs. 1, 2). The reaction cycle of topoisomerase II consists of DNA binding, DNA cleavage, DNA strand passage, and religation of the cleaved DNA (3, 4). DNA cleavage involves formation of a reversible intermediate consisting of an active site tyrosine residue that forms a phosphotyrosyl linkage with DNA. Agents targeting DNA topoisomerase II, which include important anticancer and antibacterial agents, interfere with the normal enzyme reaction cycle resulting in accumulation of DNA strand breaks with the enzyme covalently bound to DNA. These covalent complexes are reversible if drug is removed but can be converted into cytotoxic DNA damage upon collision between the com- plexes and replication forks or other DNA-tracking proteins. Consistent with generation of DNA strand breaks, homologous recombination pathways are important for cell survival following exposure to topoisomerase II poisons (5). Saccharomyces cerevisiae cells defective in rad52 as well as strains with defects in other recombination repair genes, such as rad50, mre11 , and rad54 mutants, are hypersensitive to topoisomerase II poisons (6, 7). Similar results have been observed in fission yeast mutants that are defective in DNA repair by homologous recombination (8). Other pathways for carrying out double-strand break (DSB) repair do not depend on homologous DNA as a template for repair. A critical pathway for nonhomologous repair of DSBs is the nonhomologous end-joining (NHEJ) pathway (9, 10). The essential components of NHEJ are broadly conserved among eukaryotes. Yeast and mamma- lian cells encode similar critical components, including the heterodimeric DNA-binding protein that is composed of Ku70 and Ku80 as well as a specialized DNA ligase termed ligase IV (reviewed in refs. 9, 11 – 13). There are clear differences in the components between yeast and mamma- lian cells; notably, yeast lacks the DNA-dependent protein kinase that plays a key role in many of the NHEJ reactions that occur in mammalian cells. The relative importance of NHEJ in yeast cells and metazoan organisms in repairing DNA DSBs differs Received 7/20/05; revised 4/19/06; accepted 4/28/06. Grant support: National Cancer Institute grant CA82313, core grant CA21765, and American Lebanese Syrian Associated Charities. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: The current address for V. Enriquez-Rios is Department of Chemistry and Biochemistry, University of California at Los Angeles, Los Angeles, CA 90095-1569. Requests for reprints: John L. Nitiss, Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105. Phone: 901-495-2794; Fax: 901-495-4290. E-mail: john.nitiss@stjude.org Copyright C 2006 American Association for Cancer Research. doi:10.1158/1535-7163.MCT-05-0263 1405 Mol Cancer Ther 2006;5(6). June 2006 on June 15, 2017. © 2006 American Association for Cancer Research. mct.aacrjournals.org Downloaded from