The function of DNA topoisomerases in UV-induced DNA excision repair: studies with specific inhibitors in permeabilized human fibroblasts.

Abstract

Fifteen specific inhibitors of DNA topoisomerases I and II were used to elucidate whether these enzymes participate in the excision repair of UV-induced DNA damage, monitoring DNA repair synthesis in confluent saponin-permeabilized human fibroblasts. To achieve a sufficient degree of accuracy dose--response experiments were performed, analysed by linear regression, and the concentrations at which repair activity was reduced to 50% were calculated and designated K50. Camptothecin, a specific inhibitor of topoisomerase I did not markedly diminish DNA repair synthesis. Similarly, when combined with topoisomerase II inhibitors [nalidixic acid, oxolinic acid, 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucop yra noside) (etoposide), 4'-demethylepipodophyllotoxin-thenylidene-beta-D-glucoside (teniposide), 1,4-dihydroxy-5,8-bis ((2-[(2-hydroxyethyl)amino]ethyl)amino)-9,10-anthracenedione (mitoxantrone), 5-(N-phenyl-carboxamido)-2-thiobarbituric acid (merbarone) or 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA)], it did not lower K50 values determined for topoisomerase II-specific drugs in separate experiments. The effects observed can be classified according to the mechanism of action the inhibitors exhibit. (i) Novobiocin and coumermycin, inhibitors of the ATPase subunit of topoisomerase II, completely reduced DNA repair synthesis. (ii) Inhibition of repair was also found for ethidium bromide, quinacrine and distamycin, drugs known to modify the DNA substrate by intercalation or binding to the DNA minor groove. (iii) Inhibitors acting through intercalation and, simultaneously, binding to the cleavable DNA-topoisomerase complex (m-AMSA, mitoxantrone, doxorubicin and daunorubicin) also suppressed reparative DNA synthesis. (iv) Only small effects were observed for etoposide, nalidixic acid and oxolinic acid, whereas teniposide caused marked inhibition of DNA repair synthesis. (v) Merbarone, a novel type of topoisomerase II inhibitor, blocked UV-induced DNA repair drastically. The results are best explained by assuming that in UV-irradiated human fibroblasts the 180 kDa form of topoisomerase II is the main target enzyme for inhibitors which suppressed DNA excision repair and that this isozyme is involved in steps preceding repair-specific DNA incision.