The Stereochemistry of Actinomycin Binding to DNA and Its Implications in Molecular Biology

Sobell, Henry M.

doi:10.1016/s0079-6603(08)60103-8

Cited by 148 publications

(54 citation statements)

References 48 publications

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“…Actinomycin D Does Not Inhibit Strand-Passing Activity of Topoisomerase I. Actinomycin D intercalates and unwinds DNA (28,29). The addition of actinomycin D to relaxed DNA results in the introduction of positive supercoils, and in the presence of purified topoisomerase I these positive supercoils are then relaxed.…”

Section: Resultsmentioning

confidence: 99%

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

Trask

Muller

1988

Proc. Natl. Acad. Sci. U.S.A.

147

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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...

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Section: Resultsmentioning

confidence: 99%

“…Actinomycin D inhibits transcript elongation (not initiation) by RNA polymerase, and DNA intercalation could explain the basis for inhibition of transcription in vivo (29)(30)(31) bition of transcription by the drug and the following arguments support this possibility.…”

Section: Discussionmentioning

confidence: 99%

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

Trask

Muller

1988

Proc. Natl. Acad. Sci. U.S.A.

147

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“…It is conceivable that the binding of the compounds at the restriction endonuclease recognition sites prevents binding of the enzyme or the cleavage reaction by steric hindrance. Another possible mechanism of the inhibition is the structural change of the DNA, such as a local unwinding or increased melting temperature, which is induced by the binding of the two agents [3,4]. Actinomycin D and distamycin A present at high concentrations inhibited MboI cleavage of polyoma DNA completely.…”

Section: Resultsmentioning

confidence: 99%

“…Nosikov et al reported [2] that the DNA binding compounds actinomycin D and distamycin A inhibit cleavage of DNA by a number of different restriction endonucleases. Actinomycin D intercalates between the bases of dG-dC dinucleotide residues in duplex DNA [3], whereas distamycin A binds by both electrostatic and non-ionic forces to runs of dT residues [4,5]. Digestion of phage lambda DNA showed that the two inhibitors specifically protected subsets of restriction endonuclease cleavage sites with the same recognition sequence, suggesting that base sequences contiguous to the recognition sites are important for the inhibition [5].…”

Section: Introductionmentioning

confidence: 99%

Structure at restriction endonuclease MboI cleavage sites protected by actinomycin D or distamycin A

Nilsson¹,

Skarped²,

Magnusson³

1982

FEBS Letters

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Restriction endonucleaseMboI cleavage of DNA was inhibited by actinomycin D and distamycin A. The two inhibitors protected different subsets of the 8 cleavage sites in polyoma DNA. The cleavage reactions were analyzed both in the presence of minimal inhibitory concentrations of the compounds and at higher concentrations, allowing cleavage at only 1 site/DNA molecule. The experiments showed that cleavage sites most efficiently protected by actinomycin D had putative inhibitor binding sites at a distance of 1-2 base pairs from the MboI recognition sequence. Distamycin A, in contrast, apparently has to bind immediately adjacent to the MboI recognition sequence to protect from cleavage. Polyoma

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“…Rather, the origin of the wild-type-derived nucleic acid was extracellular. Exponentially growing populations lost culturability after exposure to the DNA-interacting antibiotic actinomycin D (46). DNase treatment did not affect the culturability of the untreated cells (Fig.…”

Section: Screening For Biofilm-impaired Mutantsmentioning

confidence: 87%

DNA as an Adhesin: Bacillus cereus Requires Extracellular DNA To Form Biofilms

Vilain

Pretorius

Theron

et al. 2009

Appl Environ Microbiol

247

206

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The soil saprophyte Bacillus cereus forms biofilms at solid-liquid interfaces. The composition of the extracellular polymeric matrix is not known, but biofilms of other bacteria are encased in polysaccharides, protein, and also extracellular DNA (eDNA). A Tn917 screen for strains impaired in biofilm formation at a solid-liquid interface yielded several mutants. Three mutants deficient in the purine biosynthesis genes purA, purC, and purL were biofilm impaired, but they grew planktonically like the wild type in Luria-Bertani broth. Biofilm populations had higher purA, purC, and purL transcript ratios than planktonic cultures, as measured by real-time PCR. Laser scanning confocal microscopy (LSCM) of BacLight-stained samples indicated that there were nucleic acids in the cell-associated matrix. This eDNA could be mobilized off the biofilm into an agarose gel matrix through electrophoresis, and it was a substrate for DNase. Glass surfaces exposed to exponentially growing populations acquired a DNA-containing conditioning film, as indicated by LSCM. Planktonic exponential-phase cells released DNA into an agarose gel matrix through electrophoresis, while stationary-phase populations did not do this. DNase treatment of planktonic exponential-phase populations rendered cells more susceptible than control populations to the DNA-interacting antibiotic actinomycin D. Exponential-phase purA cells did not contain detectable eDNA, nor did they convey a DNA-containing conditioning film to the glass surface. These results indicate that exponential-phase cells of B. cereus ATCC 14579 are decorated with eDNA and that biofilm formation requires DNA as part of the extracellular polymeric matrix.

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The Stereochemistry of Actinomycin Binding to DNA and Its Implications in Molecular Biology

Cited by 148 publications

References 48 publications

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

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

Structure at restriction endonuclease MboI cleavage sites protected by actinomycin D or distamycin A

DNA as an Adhesin: Bacillus cereus Requires Extracellular DNA To Form Biofilms

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