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

Abstract

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)....

Apoptosis is induced in leishmanial cells by a novel protein kinase inhibitor withaferin A and is facilitated by apoptotic topoisomerase I–DNA complex

“…24,25 Here, we investigated the generation of such lesions by withaferin A. Leishmanial cells were permeabilized and exposed to formamidopyridine DNA glycosylase (Fpg), an enzyme that converts oxidized purines (e.g. 8-oxoguanosine) into DNA single-strand breaks.…”

Tyrosyl-DNA Phosphodiesterase as a Target for Anticancer Therapy

“…Failure to reseal this normally transient break in the DNA results in the generation of a Top1-linked DNA single-strand break that can be transformed into a prolonged doublestranded break following collision of replication forks with the drug-stabilized cleavage complex [11][12][13][14]. In a similar manner, a number of naturally occurring DNA lesions, such as strand breaks [15], abasic sites, base mismatches, and certain oxidized or modified bases [16], have also been shown in vitro to physically block the Top1 religation reaction through the misalignment the 5′-hydroxyl with the tyrosyl-DNA phosphodiester backbone. In cells, both UV irradiation- [17] and hydrogen peroxide-induced [18] DNA damage have also been shown to trap Top1 cleavage complexes.…”

Interaction of Human Nuclear Topoisomerase I with Guanosine Quartet-forming and Guanosine-rich Single-stranded DNA and RNA Oligonucleotides

“…Top1 inhibitors, such as camptothecin and its derivatives, selectively inhibit eukaryotic top1 by reversibly preventing the religation step of the enzyme-mediated DNA nicking-closing reaction (24,25). Dissociation of the DNA strand 3Ј from the cleavage site, due to the presence of a nick, gap, or single-stranded branch in the vicinity, prevents the religation step of top1 catalytic reaction (26) and leads to irreversible cleavage complexes (27). Such substrates, commonly referred to as "suicide substrates," have been very useful in the study of the top1 catalytic cycle because they can be used to monitor, under single turnover conditions, the top1-mediated DNA cleavage step (28,29).…”

“…A partially singlestranded 18-mer ODN, TH-18 (Fig. 1B), containing a high affinity site (26,35) (Fig. 1A) was used as a "suicide substrate" (36).…”