New technique for uncoupling the cleavage and religation reactions of Eukaryotic Topoisomerase I.

Svejstrup, Jesper Q.; Christiansen, Karl O.; Gromova, Irina; Andersen, Anders H.; Westergaard, Ole

doi:10.1016/0022-2836(91)90503-x

Cited by 151 publications

(131 citation statements)

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“…Top1 suicide (or aborted product) is a very efficient donor for the religation of an exogenous DNA duplex bearing a 5Ј-hydroxyl end (Figs. 3-5) (12,13,18,22,24,27,40). Religation of blunt-ended DNA duplex molecules is not influenced by the terminal sequence of the acceptor (13).…”

Section: Discussionmentioning

confidence: 99%

“…We recently reported that top1 can be trapped irreversibly by common DNA endogenous lesions such as DNA mismatches and abasic sites (21). Previous studies also reported that nicks in the vicinity of the top1 cleavage site were able to generate double-strand breaks, leading to suicide or aborted products, where covalently bound top1 is able to religate various nonhomologous acceptors bearing a 5Ј-hydroxyl terminus and thus participates into illegitimate recombination (10,12,13,(22)(23)(24).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).…”

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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

164

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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Section: Discussionmentioning

confidence: 99%

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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

164

118

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“…However, the relative contributions of this interaction to noncovalent vs. covalent binding would appear to differ for the cellular and viral enzymes. Initial conclusions regarding the 'minimal' DNA duplex requirements for suicide cleavage by cellular topoisomerase I (13) have been revised, with the end result now being that covalent adduct formation requires a duplex segment of DNA extending 1 I-bp downstream of the site of strand scission (11 (4). Rather, as shown herein, the influence of the downstream region on the vaccinia enzyme is on precleavage binding.…”

Section: -Mermentioning

confidence: 99%

Requirements for noncovalent binding of vaccinia topoisomerase I to duplex DNA

Sekiguchi

Shuman

1994

Nucl Acids Res

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“…To investigate the mechanism of topoisomerase I inhibition by NB-506, we first analyzed its effects on the re-ligation reaction. It has been reported that the DNA cleavage reaction of topoisomerase I is inhibited by high concentrations of NaCl; however, the re-ligation reaction is not affected by NaCl concentrations up to 3 M (Svejstrup et al, 1991). Based on this feature, the re-ligation rate of cleaved DNA was evaluated in the presence of 0.35 M NaCl after the formation of cleavable complex by NB-506 and camptothecin.…”

Section: Induction Of Dna Cleavage and Inhibition Of Religation Reactmentioning

confidence: 99%

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

et al. 1998

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New technique for uncoupling the cleavage and religation reactions of Eukaryotic Topoisomerase I.

Cited by 151 publications

References 29 publications

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

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

Requirements for noncovalent binding of vaccinia topoisomerase I to duplex DNA

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

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