A high affinity topoisomerase I binding sequence is clustered at DNAase I hypersensitive sites in tetrahymena R-chromatin

Bonven, Bjarne; Gocke, Elmar; Westergaard, Ole

doi:10.1016/s0092-8674(85)80027-1

Cited by 256 publications

(161 citation statements)

References 40 publications

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“…2) was efficiently cleaved by human top1 at the expected site in the presence of camptothecin (7,12). DNA cleavage was between the T and A bases (caret in Fig.…”

Section: Accumulation Of Top1-mediated Dna Cleavage Complexes By Bap mentioning

confidence: 98%

“…We synthesized oligonucleotides similar in sequence to those used to determine the crystal structures of top1-DNA complexes (7,8). This sequence is derived from Tetrahymena R-chromatin at a previously identified and wellcharacterized top1 cleavage site (12)(13)(14). To obtain DNA oligonucleotides with intercalated adducts at selected positions relative to the normal top1 cleavage site, single trans-opened benzo[a]pyrene (BaP) diol epoxide (DE-2, benzylic 7-hydroxyl group and epoxide oxygen are trans) adducts at N 6 of dA ( Fig.…”

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confidence: 99%

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Position-specific trapping of topoisomerase I–DNA cleavage complexes by intercalated benzo[ a ]- pyrene diol epoxide adducts at the 6-amino group of adenine

Pommier

Laco

Kohlhagen

et al. 2000

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

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DNA topoisomerase I (top1) is the target of potent anticancer agents, including camptothecins and DNA intercalators, which reversibly stabilize (trap) top1 catalytic intermediates (cleavage complexes). The aim of the present study was to define the structural relationship between the site(s) of covalently bound intercalating agents, whose solution conformations in DNA are known, and the site(s) of top1 cleavage. Two diastereomeric pairs of oligonucleotide 22-mers, derived from a sequence used to determine the crystal structure of top1-DNA complexes, were synthesized. One pair contained either a trans-opened 10R-or 10S-benzo[a]pyrene 7,8-diol 9,10-epoxide adduct at the N 6 -amino group of a central 2 -deoxyadenosine residue in the scissile strand, and the other pair contained the same two adducts in the nonscissile strand. These adducts were derived from the (؉)-(7R,8S,9S,10R)-and (؊)-(7S,8R,9R,10S)-7,8-diol 9,10-epoxides in which the benzylic 7-hydroxyl group and the epoxide oxygen are trans. On the basis of analogy with known solution conformations of duplex oligonucleotides containing these adducts, we conclude that top1 cleavage complexes are trapped when the hydrocarbon adduct is intercalated between the base pairs flanking a preexisting top1 cleavage site, or between the base pairs immediately downstream (3 relative to the scissile strand) from this site. We propose a model with the ؉1 base rotated out of the duplex, and in which the intercalated adduct prevents religation of the corresponding nucleotide at the 5 end of the cleaved DNA. These results suggest mechanisms whereby intercalating agents interfere with the normal function of human top1. D NA topoisomerase I (top1) is an essential enzyme in higher eukaryotes (1, 2). It can relax DNA supercoiling and relieve torsional strain during DNA processing, including replication, transcription, and repair. It can also perform intermolecular religation leading to DNA recombinations (3, 4). The catalytic intermediate of top1 is a cleavage complex in which a tyrosine (Tyr-723 for human top1) in the enzyme attacks a DNA phosphodiester and forms a covalent bond to the phosphorus at the 3Ј side of the cleavage site while a 5Ј-hydroxyl is generated at the other side (1, 2). The reaction occurs with short duplex oligodeoxynucleotides (5, 6), as evidenced by recent crystal structures of top1-DNA complexes (7,8).top1 is the cellular target of several anticancer drugs, including the camptothecins, which have recently been approved by the Food and Drug Administration for the treatment of colon and ovarian carcinomas. The anticancer activity of these drugs results from the trapping of top1 cleavage complexes such that the enzyme is reversibly inactivated as it cleaves DNA (9-11). These agents are often referred to as ''top1 poisons.'' They represent an exemplary case of pharmacological interference whereby the drug stabilizes the interactions of two macromolecules (i.e., top1 and DNA) by formation of a ternary complex. Because of the clinical efficacy of camptothecins, no...

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“…2) was efficiently cleaved by human top1 at the expected site in the presence of camptothecin (7,12). DNA cleavage was between the T and A bases (caret in Fig.…”

Section: Accumulation Of Top1-mediated Dna Cleavage Complexes By Bap mentioning

confidence: 98%

mentioning

confidence: 99%

Position-specific trapping of topoisomerase I–DNA cleavage complexes by intercalated benzo[ a ]- pyrene diol epoxide adducts at the 6-amino group of adenine

Pommier

Laco

Kohlhagen

et al. 2000

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

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“…In this study, we used purified mammalian top1 and DNA oligonucleotides containing a unique top1 cleavage site derived from the rDNA sequence of Tetrahymena (25,26) to study top1-mediated DNA damage and recombination induced in DNA substrates containing nicks or gaps in the non-scissile strand (i.e. opposite to the cleaved strand).…”

mentioning

confidence: 99%

Trapping of Mammalian Topoisomerase I and Recombinations Induced by Damaged DNA Containing Nicks or Gaps

Pourquier

Pilon

Kohlhagen

et al. 1997

Journal of Biological Chemistry

163

118

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We used purified mammalian topoisomerases I (top1) and oligonucleotides containing a unique top1 cleavage site to study top1-mediated cleavage and recombination in the presence of nicks and short gaps mimicking DNA damage. In general, top1 cleavage was not induced opposite to the nicks, and nicks upstream from the top1 cleavage site suppressed top1 activity. Irreversible top1 cleavage complexes ("suicide products" or "aborted complexes") were produced in DNA containing nicks or short gaps just opposite to the normal top1 cleavage site. Camptothecin enhanced the formation of the aborted top1 complexes only for nicks downstream from the cleavage site. These aborted (suicide) complexes can mediate DNA recombination and promote illegitimate recombination by catalyzing the ligation of nonhomologous DNA fragments (acceptors). We report for the first time that top1-mediated recombination is greatly enhanced by the presence of a phosphate at the 5 terminus of the top1 aborted complex (donor DNA). By contrast, phosphorylation of the 3 terminus of the gap did not affect recombination. At concentrations that strongly enhanced inhibition of intramolecular religation, resulting in an increase of top1 cleavable complexes, camptothecin did not reduce recombination (intermolecular religation). Nicks or gaps with 5 -phosphate termini would be expected to be produced directly by ionizing radiations or by processing of abasic sites and DNA lesions induced by carcinogens or drugs used in cancer chemotherapy. Thus, these results further demonstrate that DNA damage can efficiently trap top1-cleavable complexes and enhance top1-mediated DNA recombination.DNA rearrangements such as deletions, translocations, inversions, or gene amplifications are involved in genetic diseases and carcinogenesis (1-4). These modifications are based on the cell's ability to promote recombinations. This process can be initiated after exposure to exogenous DNA damaging agents such as ionizing radiations, environmental carcinogens, and drugs used in cancer chemotherapy (5). Even though their molecular mechanisms are not yet fully understood, two types of recombinations have been described depending on the homology of the DNA molecules joined. In eukaryotes, the nonhomologous (or illegitimate) recombination occurs more frequently than the homologous (or legitimate) recombination (6).Topoisomerases are ubiquitous enzymes involved in multiple processes including replication, transcription, chromosome segregation, and recombinations (7,8). DNA topoisomerase I (top1) 1 acts as a monomer and creates transient DNA singlestrand breaks via the formation of a covalent bond between the 3Ј-phosphate of the cleaved strand and a tyrosine residue of the enzyme. Under physiological conditions, these intermediates are referred to as cleavable complexes. The cleavable complexes are readily reversible by top1-mediated religation of the 5Ј-hydroxyl termini (7,9,10).In mammalian cells, top1 is involved in illegitimate recombination in vitro (11-15) and probably in vivo (16 -18)....

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“…A synthetic oligonucleotide containing Tetrahymena topoisomerase I binding sequence (Bonven et al, 1985) was inserted into the HindIII-EcoR I site of pBR322. Plasmid DNA was digested with BamHI and 3Ј-end-labeled with [␣-32 P]-dCTP and the Klenow fragment of DNA polymerase I. Enzymatic digestion with EcoRI yielded 2 single-end-labeled DNA fragments; the 258-bp fragment was isolated from 1.5% agarose gel.…”

Section: Dna Substratesmentioning

confidence: 99%

“…A topoisomerase Imediated DNA cleavage assay was performed using a 3Ј-endlabeled 258-bp DNA fragment containing a Tetrahymena topoisomerase I binding sequence (Bonven et al, 1985). Because topoisomerase I-mediated DNA cleavage generates a 5Ј-OH DNA terminus instead of the 5Ј-P terminus produced by the MaxamGilbert chemical reaction (Jaxel et al, 1988), the marker fragments were treated with a bacterial alkaline phosphatase after the Maxam-Gilbert chemical reaction.…”

Section: Characteristics Of the Topoisomerase I-mediated Dna Cleavagementioning

confidence: 99%

Sequence-selective DNA cleavage by a topoisomerase I poison, NB-506

et al. 1998

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A high affinity topoisomerase I binding sequence is clustered at DNAase I hypersensitive sites in tetrahymena R-chromatin

Cited by 256 publications

References 40 publications

Position-specific trapping of topoisomerase I–DNA cleavage complexes by intercalated benzo[ a ]- pyrene diol epoxide adducts at the 6-amino group of adenine

Position-specific trapping of topoisomerase I–DNA cleavage complexes by intercalated benzo[ a ]- pyrene diol epoxide adducts at the 6-amino group of adenine

Trapping of Mammalian Topoisomerase I and Recombinations Induced by Damaged DNA Containing Nicks or Gaps

Sequence-selective DNA cleavage by a topoisomerase I poison, NB-506

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