A number of DNA minor groove-binding ligands (MGBLs) are known to exhibit antitumor and antimicrobial activities. We show that DNA topoisomerase (Topo) I may be a pharmacological target of MGBLs. In the presence of calf thymus Topo I, MGBLs induced limited but highly specific single-strand DNA breaks. The 3' ends of the broken DNA strands are covalently linked to Topo I polypeptides. Proteinlinked DNA breaks are readily reversed by a brief heating to 65°C or the addition of 0.5 M NaCl. These results suggest that MGBLs, like camptothecin, abort Topo I reactions by trapping reversible deavable complexes. The sites of cleavage induced by MGBLs are distinctly different from those induced by camptothecin. Two of the major cleavage sites have been sequenced and shown to be highly A+T-rich, suggesting the possible involvement of a Topo I-drug-DNA ternary complex at the sites of deavage. Different MGBLs also exhibit varying effciency in inducing Topo I-cleavable complexes, and the order of efficiency is as follows: Hoechst 33342 and 33258 >> distamycin A > berenil > netropsin. The lack of correlation between DNA binding and cleavage efficiency suggests that, in addition to binding to the minor grooves ofDNA, MGBLs must also interact with Topo I in trapping Topo I-cleavable complexes.Many DNA-binding (5, 6). Their modes of interaction with DNA have been studied extensively (reviewed in refs. 7 and 8). Some of the most commonly studied minor groove-binding compounds, such as distamycin A, netropsin, and the bisbenzimidazole dyes (Hoechst dyes) [e.g., 2'-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi-lH-benzimidazole trihydrochloride trihydrate (Hoechst 33342) and 2'-(4-hydroxyphenyl)-5-(4-methyl-1-piperizinyl)-2,5'-bi-lH-benzimidazole trihydrochloride pentahydrate (Hoechst 33258)] are known to bind to the minor groove of DNA with A+T specificity and to cause widening of the minor grooves (7, 8). Despite extensive investigation into the DNA-binding mode of these compounds, the cellular targets of these compounds other than DNA have not been identified.Most DNA minor groove-binding ligands (MGBLs) are positively charged (see Fig. 1 MATERUILS AND METHODS Materials. Hoechst 33342 and 33258, DAPI (4',6-diamidino-2-phenylindole), berenil, and distamycin A were purchased from Sigma; netropsin was from Boehringer Mannheim. All compounds were dissolved in 10 mM dimethyl sulfoxide and kept frozen in aliquots at -20°C. DNA Topos I and II were purified from calf thymus glands by using published procedures (14, 15). YEpG is a derivative of YEP24, which has the regulated yeast GALI promoter inserted between the BamHI and Sal I sites ofthe tetracyclineresistance gene (16). The Sequenase kit was purchased from United States Biochemical.DNA Topos I and H Cleavage Assays. DNA Topos I and II cleavage assays were done as described (17, 18). The procedure for end-labeling ofplasmid DNA has been described (19). Mapping and Sequencing the Cleavage Sites. The mapping of cleavage sites induced by Hoechst 33342 and 33258 was done ...

Hydrogen peroxide (H 2 O 2 ), a reactive oxygen species (ROS), is known to induce oxidative stress and apoptosis. U937 cells treated with H 2 O 2 were shown to produce high molecular weight (HMW) DNA fragments ∼50 to 100 kb in size in <1 min. The formation of these HMW DNA fragments is reversible and shown to be mediated by DNA topoisomerase II (TOP2). Following this initial event, formation of irreversible HMW DNA fragments and nucleosomal ladders occurs. Our results thus demonstrate a potential role of TOP2 in oxidative damage of DNA and apoptotic cell death.

Several 2'-aryl-5-substituted-2,5'bi-1H-benzimidazole derivatives were synthesized and evaluated as topoisomerase I poisons and for their cytotoxicity toward the human lymphoblast cell line RPMI 8402. This study focused on 18 2,5'-bi-1H-benzimidazole derivatives which contained either a 5-cyano, a 5-(aminocarbonyl), or a 5-(4-methylpiperazinyl) group. Among these bibenzimidazoles, the pharmacological activity of 2'-phenyl derivatives and the influence of the different positional isomers of either a 2'-tolyl group or a 2'-naphthyl moiety on cytotoxicity and topoisomerase I inhibitory activity were determined.

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Spectroscopic, calorimetric, DNA cleavage, electrophoretic, and computer modeling techniques have been employed to characterize the DNA binding and topoisomerase poisoning properties of three protoberberine analogs, 8-desmethylcoralyne (DMC), 5,6-dihydro-8-desmethylcoralyne (DHDMC), and palmatine, which differ in the chemical structures of their B- and/or D-rings. DNA topoisomerase-mediated cleavage assays revealed that these compounds were unable to poison mammalian type II topoisomerase. By contrast, the three protoberberine analogs poisoned human topoisomerase I according to the following hierarchy: DHDMC > DMC > palmatine. DNA binding by all three protoberberine analogs induced negative flow linear dichroism signals as well as unwinding of the host duplex. These two observations are consistent with an intercalative mode of protoberberine binding to duplex DNA. However, a comparison of the DNA binding properties for DMC and DHDMC, which differ only by the state of saturation at the 5,6 positions of the B-ring, revealed that the protoberberine analogs do not "behave" like classic DNA intercalators. Specifically, saturation of the 5-6 double bond in the B-ring of DMC, thereby converting it to the DHDMC molecule, was associated with enhanced DNA unwinding as well as a reversal of DNA binding preference from a DNA duplex with an inaccessible or occluded minor groove {poly[d(G-C)]2} to DNA duplexes with accessible or unobstructed minor grooves {poly[d(A-T)]2 and poly[d(I-C)]2}. In addition, a comparison of the DNA binding properties for DHDMC and palmatine revealed that transferring the 11-methoxy moiety on the D-ring of DHDMC to the 9 position, thereby converting it to palmatine, was associated with a reduction in binding affinity for both duplexes with unobstructed minor grooves as well as for duplexes with occluded minor grooves. These DNA binding properties are consistent with a "mixed-mode" DNA binding model for protoberberines in which a portion of the ligand molecule intercalates into the double helix, while the nonintercalated portion of the ligand molecule protrudes into the minor groove of the host duplex, where it is thereby available for interactions with atoms lining the floor and/or walls of the minor groove. Furthermore, saturation at the 5,6 positions of the B-ring, which causes the A-ring to be tilted relative to the plane formed by rings C and D, appears to stabilize the interaction between the host duplex and the minor groove-directed portion of the protoberberine ligand. Computer modeling studies on the DHDMC-poly[d(A-T)]2 complex suggest that this interaction may involve van der Waals contacts between the ligand A-ring and backbone sugar atoms lining the minor groove of the host duplex. The hierarchy of topoisomerase I poisoning noted above suggests that this minor groove-directed interaction may play an important role in topoisomerase I poisoning by protoberberine analogs. In the aggregate, our results presented here, coupled with the recent demonstration of topoisomerase I poisoning by minor ...