Genetic evidence for involvement of two distinct nonhomologous end-joining pathways in repair of topoisomerase II-mediated DNA damage.
In vertebrate cells, DNA double-strand breaks are efficiently repaired by homologous recombination or nonhomologous end-joining (NHEJ). The latter pathway relies on Ku (the Ku70/Ku86 heterodimer), DNA-PKcs, Artemis, Xrcc4, and DNA ligase IV (Lig4). Here, we show that a human pre-B cell line nullizygous for Lig4 exhibits hypersensitivity to topoisomerase II (Top2) inhibitors, demonstrating a crucial role for the NHEJ pathway in repair of Top2-induced DNA damage in vertebrates. We also show that in the chicken DT40 cell line, all NHEJ mutants (i.e., Ku70-, Lig4-, and DNA-PKcs-null cells) are equally hypersensitive to the Top2 inhibitor ICRF-193, indicating that the drug-induced damage is repaired by NHEJ involving DNA-PKcs. Intriguingly, however, DNA-PKcs-null cells display considerably less severe phenotype than other NHEJ mutants in terms of hypersensitivity to VP-16, a Top2 poison that stabilizes cleavable complexes. The results indicate that two distinct NHEJ pathways, involving or not involving DNA-PKcs, are important for the repair of VP-16-induced DNA damage, providing additional evidence for the biological relevance of DNA-PKcs-independent NHEJ. Our results provide significant insights into the mechanisms of repair of Top2-mediated DNA damage, with implications for chemotherapy involving Top2 inhibitors.
Adachi
Noritaka
N
Kihara Institute for Biological Research, Graduate School of Integrated Science, Yokohama City University, Totsuka-ku, Yokohama 244-0813, Japan. nadachi@yokohama-cu.ac.jp
Iiizumi
Susumu
S
So
Sairei
S
Koyama
Hideki
H
D000818:Animals; D002460:Cell Line; D002645:Chickens; D004249:DNA Damage; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000235:genetics; D004260:DNA Repair / Q000187:drug effects / Q000235:genetics*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D054659:Diketopiperazines; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005047:Etoposide / Q000494:pharmacology; D006801:Humans; D010641:Phenotype; D010879:Piperazines / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics*; D059005:Topoisomerase II Inhibitors
D054659:Diketopiperazines; D004791:Enzyme Inhibitors; C000607893:LIG4 protein, human; D010879:Piperazines; D059005:Topoisomerase II Inhibitors; C070899:4,4'-(1,2-dimethyl-1,2-ethanediyl)bis-2,6-piperazinedione; D005047:Etoposide; D017346:Protein Serine-Threonine Kinases; D004250:DNA Topoisomerases, Type II; D011088:DNA Ligases; D000072481:DNA Ligase ATP