Stabilization of type I topoisomerase-DNA covalent complexes by actinomycin D.

Abstract

The activity of the endogenous DNA topoisomerase type I (EC 5.99.1.2) can be quantified in situ by determining how efficiently the enzyme is trapped in a covalent complex with DNA upon lysis of nuclei with detergents. In this way, we can measure relative levels of topoisomerase binding to DNA at native sites in chromatin. Since the majority of topoisomerase I is localized in the nucleolus at rRNA genes, we have evaluated how low levels of actinomycin D, which terminate transcription of rRNA genes, affect the activity of topoisomerase I. In vivo, as well as in vitro with purified topoisomerase I, we have found that drug treatment extends the half-life of the covalent topoisomerase-DNA complex. Actinomycin D stabilizes the nicked intermediate in the cleavage and resealing reaction but otherwise does not significantly alter the strand-passing ability of topoisomerase I. Sequence-specific cleavages by topoisomerase I were stimulated by actinomycin D at identical sequences recognized by the enzyme in the absence of drug. The localization of topoisomerase I in the nucleolus, coupled with the observation that transcription in this organelle is highly sensitive to actinomycin D and camptothecin treatment, leads us to propose that topoisomerase I contributes to actinomycin D inhibition of transcription.A number of reports have suggested that type I DNA topoisomerase (topoisomerase I; EC 5.99.1.2) is involved in transcription based on its association with actively transcribed genes in chromatin (1-6). This association is clearly evident for rRNA genes in animals, yeast, and Tetrahymena (2,3,7). The enzyme is acting catalytically at genes characterized by a high rate of transcription and the heaviest enrichment of topoisomerase I is seen cytologically within the nucleolus (2). Topoisomerase I makes a transient singlestrand break in the sugar-phosphate backbone of DNA (for reviews, see refs. 8 and 9), which introduces a site of rotational freedom in the template; thus, topoisomerase action may provide a swivel point to facilitate entry and/or progression of the bulky transcriptional apparatus. Alternatively, when nascent RNA chains are hybridized to the one strand of template, the topology changes and topoisomerase I may be required to return to (or adjust) the topological ground state. Studies of yeast topoisomerase I mutants suggest that topoisomerase I is not an essential gene (10, 11); however, it seems that topoisomerase II (EC 5.99.1.3) is complementing the defect since topoisomerase II (like topoisomerase I) has been associated with transcriptionally active regions in chromatin (ref. 12; M.T.M. and V. Mehta, unpublished data) and topoisomerase I and II double mutants display a defect in rRNA transcription (7).A better understanding of the involvement of topoisomerase I in transcription can be obtained by analyzing the activity of topoisomerase I catalyzed reactions in a chromosomal setting. The covalent intermediate is a functional reaction intermediate in the process of breaking and rejoining the DNA substra...

Actinomycin D Stimulates the Transcription of rRNA Minigenes Transfected into Mouse Cells. Implications for the in vivo Hypersensitivity of rRNA Gene Transcription

“…In all cases, 1-2 h after addition of 0.01-0.08 pg/ml actinomycin D we observe, a complete absence of full-length transcripts, and an accumulation of abortive incomplete transcripts. These results show that the stimulation of transcription initiation by actinomycin D is superimposed upon a blockage of transcription elongation, a finding in agreement with earlier results of Fetherston et al [13] showing a blockage of rRNA gene transcription elongation by actinomycin D. Our results are also in accord with the established inhibition of DNA topoisomerase I by actinomycin D [19] and indirectly with those on the blockage of transcription elongation by the DNA topoisomerase I inhibitor camptothecin [22]. All these observations strengthen the view that the actinomycin D blockage of rRNA gene transcription elongation is due primarily to enhanced transcription-driven torsional tension [21, 24, 251 rather than to a selective interaction of the drug with a particular transcribed rDNA sequence.…”

“…In vitro studies by Trask and Muller [19] showed that low concentrations (0.1 pg/ml) of actinomycin D stabilize the covalent topoisomerase-I-DNA complexes, thus inhibiting topoisomerase I activity and contributing to the inhibition of rRNA gene transcription elongation. The involvement of topoisomerase I in rRNA gene transcription stressed the possible role of DNA supercoiling in transcription control [20, 211, a factor whose importance has been documented in several studies including those on the transcription of eukaryotic rRNA genes either in vitro [22] or in cellular [23-251 systems. Further understanding of the hypersensitivity of rRNA gene transcription to actinomycin D has been seriously hampered by the observations showing that neither transient transfection [26], nor in vitro [6] transcription systems, could reproduce the preferential in vivo inhibition of rRNA gene transcription by low doses of the drug 161.…”

Proteomic Analysis of Arginine Methylation Sites in Human Cells Reveals Dynamic Regulation During Transcriptional Arrest

“…To further investigate the biological implications of MMA sites in RNA metabolism, we performed a temporal proteomics experiment aimed at mapping regulated methylation sites upon transcriptional arrest by Actinomycin D (ActD). ActD is a widely used transcriptional inhibitor that intercalates into G-C rich DNA regions and prevents the progression of RNA polymerase (17). Interestingly, our analysis identifies several MMA sites regulated upon inhibition of transcription, whereas no regulation is observed for the corresponding di-methylation or protein turnover.…”

Phase I and pharmacokinetic study of DACA (XR5000): a novel inhibitor of topoisomerase I and II

“…Until recently, actinomycin D was the only single agent with demonstrable activity against topoisomerases I and II (Tewey et al, 1984;Trask and Muller, 1988). In a phase I trial of intoplicine, a 7-H-benzo[e]pyridol [4,3-b]-indole derivative that also has activity against topoisomerases I and II, hepatotoxicity was dose-limiting (Abigerges et al, 1996).…”