Exposure to camptothecin breaks leading and lagging strand simian virus 40 DNA replication forks

doi:10.1016/0006-291X(90)91684-K

Biochemical and Biophysical Research Communications

Volume 168, Issue 1, 16 April 1990, Pages 135-140

https://doi.org/10.1016/0006-291X(90)91684-K Get rights and content

Abstract

To better understand aberrant simian virus 40 DNA replication intermediates produced by exposure of infected cells to the anticancer drug camptothecin, we compared them to forms produced by S1 nuclease digestion of normal viral replication intermediates. All of the major forms were identical in both cases. Thus the aberrant viral replicating forms in camptothecin-treated cells result from DNA strand breaks at replication forks. Linear simian virus 40 forms which are produced by camptothecin exposure during viral replication were identified as detached DNA replication bubbles. This indicates that double strand DNA breaks caused by camptothecin-topoisomerase I complexes occur at both leading and lagging strand replication forks $in vivo$ .

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Cited by (39)

Recombinogenic flap ligation mediated by human topoisomerase I
2003, Journal of Molecular Biology
Citation Excerpt :
topo I action per se rarely introduces damage to the DNA backbone. However, in the presence of chemotherapeutica, such as camptothecin or its derivatives, a high frequency of topo I-bound DSBs is induced in the genome, making flap ligation a relevant factor in relation to cancer treatment.18–20 Cheng & Shuman suggested flap ligation to be particularly important during the transition between check-point arrest and apoptosis induced by the treatment with camptothecins, since under these conditions DNase II-like activities generate a high level of free 5′-hydroxyl ends that serve as acceptors for topo I-mediated ligation.21
Aberration of eukaryotic topoisomerase I catalysis leads to potentially recombinogenic pathways by allowing the joining of heterologous DNA strands. Recently, a new ligation pathway (flap ligation) was presented for vaccinia virus topoisomerase I, in which blunt end cleavage complexes ligate the recessed end of duplex acceptors having a single-stranded 3′-tail. This reaction was suggested to play an important role in the repair of topoisomerase I-induced DNA double-strand breaks. Here, we characterize flap ligation mediated by human topoisomerase I. We demonstrate that cleavage complexes containing the enzyme at a blunt end allow invasion of a 3′-acceptor tail matching the scissile strand of the donor, which facilitates ligation of the recessed 5′-hydroxyl end. However, the reaction was strictly dependent on the length of double-stranded DNA of the donor complexes, and longer stretches of base-pairing inhibited strand invasion. The stabilization of the DNA helix was most probably provided by the covalently bound enzyme itself, since deleting the N-terminal domain of human topoisomerase I stimulated flap ligation. We suggest that stabilization of the DNA duplex upon enzyme binding may play an important role during normal topoisomerase I catalysis by preventing undesired strand transfer reactions. For flap ligation to function in a repair pathway, factors other than topoisomerase I, such as helicases, would be necessary to unwind the DNA duplex and allow strand invasion.
A phase I study of sequential irinotecan and 5-fluorouracil/leucovorin
2002, Annals of Oncology
Citation Excerpt :
CPT-11 is converted in vivo to SN-38, which is 1000 times more potent than the parent drug [7–9]. SN-38 targets topoisomerase I, covalently stabilizing the enzyme–DNA complexes [10], resulting in strand breakage and subsequent cytotoxicity [11–14]. Phase II trials have shown that CPT-11 has activity in a wide spectrum of human neoplasms [15–22], and recent phase III studies indicate that CPT-11 can increase survival in patients with 5-fluorouracil (5-FU)-resistant metastatic colorectal cancer [23, 24].
Irinotecan (CPT-11) and 5-fluorouracil (5-FU)/leucovorin are active agents in colorectal cancer. A sequence-dependent synergism of SN-38 followed by 5-FU/leucovorin in vitro led us to conduct a phase I trial of CPT-11 followed by 5-FU/leucovorin to determine the maximum tolerated dose (MTD) and toxicities of this regimen and to obtain preliminary indications of its activity in patients with advanced solid tumors.
Fifty-six patients were enrolled in sequential cohorts to receive escalating doses of CPT-11 (90 min infusion) on day 1, followed by leucovorin 20 mg/m² (intravenous push) and 5-FU (90 min infusion) on days 2–5 of each 21-day cycle.
A total of 347 treatment cycles (median 4, range 1–25) were administered. Dose-limiting toxicities were diarrhea, neutropenia and fatigue. Nine patients with colorectal cancer and one with gastric cancer had partial or minor responses. Eight of the 10 had prior chemotherapy.
CPT-11 and 5-FU/leucovorin, as constituents of this novel mechanism-based schedule, have promising activity in patients who have received prior chemotherapy. The recommended phase II/III starting doses are CPT-11 275 mg/m² over 90 min on day 1, and 5-FU 400 mg/m² plus leucovorin 20 mg/m² on days 2–5 every 21 days. This combination can be administered safely to this schedule if there is strict adherence to the 90 min infusion time for both CPT-11 and 5-FU.
S phase and G<inf>2</inf> arrests induced by topoisomerase I poisons are dependent on ATR kinase function
2002, Journal of Biological Chemistry
Citation Excerpt :
In contrast, the G2 arrest was more persistent in the presence of normal ATR function (Fig. 1A, 48 h). Prolonged exposure to topo I poisons results in slowing of DNA replication (32-34), presumably because of activation of the S phase checkpoint by replication-induced conversion of the stabilized DNA-topo I complexes into frank DNA ds breaks (26-29). To evaluate the possibility that ATR might also function as part of this S phase checkpoint, we pretreated cells for 48 h with doxycycline (to inactivate ATR function) or diluent, then exposed them to 125 nm TPT for 8 h in the continued presence or absence of doxycycline.
ATR, a human phosphatidylinositol 3-kinase-related kinase, is an important component of the cellular response to DNA damage. In the present study, we evaluated the role of ATR in modulating the response of cells to S phase-associated DNA double-stranded breaks induced by topoisomerase poisons. Prolonged exposure to low doses of the topoisomerase I poison topotecan (TPT) resulted in S phase slowing because of diminished DNA synthesis at late-firing replicons. In contrast, brief TPT exposure, as well as prolonged exposure to the topoisomerase II poison etoposide, resulted in subsequent G₂ arrest. These responses were associated with phosphorylation of the checkpoint kinase Chk1. The cell cycle responses and phosphorylation of Chk1 were markedly diminished by forced overexpression of a dominant negative, kinase-inactive allele of ATR. In contrast, deficiency of the related kinase ATM had no effect on these events. The loss of ATR-dependent checkpoint function sensitized GM847 human fibroblasts to the cytotoxic effects of the topoisomerase I poisons TPT and 7-ethyl-10-hydroxycamptothecin, as assessed by inhibition of colony formation, increased trypan blue uptake, and development of apoptotic morphological changes. Expression of kdATR also sensitized GM847 cells to the cytotoxic effects of prolonged low dose etoposide and doxorubicin, albeit to a smaller extent. Collectively, these results not only suggest that ATR is important in responding to the replication-associated DNA damage from topoisomerase poisons, but also support the view that ATM and ATR have unique roles in activating the downstream kinases that participate in cell cycle checkpoints.
Expression of human topoisomerase I with a partial deletion of the linker region yields monomeric and dimeric enzymes that respond differently to camptothecin
2000, Journal of Biological Chemistry
Human topoisomerase I is a 765-residue protein composed of four major domains as follows: the unconserved and highly charged NH₂-terminal domain, a conserved core domain, the positively charged linker region, and the highly conserved COOH-terminal domain containing the active site tyrosine. Previous studies of the domain structure revealed that near full topoisomerase I activity can be reconstituted in vitro by fragment complementation between recombinant polypeptides approximating the core and COOH-terminal domains. Here we demonstrate that deletion of linker residues Asp⁶⁶⁰ to Lys⁶⁸⁸ yields an active enzyme (topo70ΔL) that purifies as both a monomer and a dimer. The dimer is shown to result from domain swapping involving the COOH-terminal and core domains of the two subunits. The monomeric form is insensitive to the anti-tumor agent camptothecin and distributive during in vitro plasmid relaxation assays, whereas the dimeric form is camptothecin-sensitive and processive. However, the addition of camptothecin to enzyme/DNA mixtures causes enhancement of SDS-induced breakage by both monomeric and dimeric forms of the mutant enzyme. The similarity of the dimeric form to the wild type enzyme suggests that some structural feature of the dimer is providing a surrogate linker. Yeast cells expressing topo70ΔL were found to be insensitive to camptothecin.
Mechanism of action of eukaryotic DNA topoisomerase I and drugs targeted to the enzyme
1998, Biochimica et Biophysica Acta - Gene Structure and Expression
DNA topoisomerase I is essential for cellular metabolism and survival. It is also the target of a novel class of anticancer drugs active against previously refractory solid tumors, the camptothecins. The present review describes the topoisomerase I catalytic mechanisms with particular emphasis on the cleavage complex that represents the enzyme’s catalytic intermediate and the site of action for camptothecins. Roles of topoisomerase I in DNA replication, transcription and recombination are also reviewed. Because of the importance of topoisomerase I as a chemotherapeutic target, we review the mechanisms of action of camptothecins and the other topoisomerase I inhibitors identified to date.
Cell death induced by topoisomerase-targeted drugs: More questions than answers
1998, Biochimica et Biophysica Acta - Gene Structure and Expression
Chemotherapeutic agents that target topoisomerase I and II set into motion a series of biochemical changes that culminate in cell death, but only under some conditions. The realization that stabilization of covalent topoisomerase–DNA complexes is not sufficient to insure cell death has prompted investigators to examine various aspects of the drug-induced death process itself. Several discrete steps along this pathway have been identified, including (a) the processing of stabilized cleavage complexes into frank DNA strand breaks; (b) sensing of the DNA damage, leading to activation of stress-associated signaling pathways and cell cycle arrest; and (c) activation of a preexisting group of enzymes and enzyme precursors, typified by the cysteine-dependent aspartate-directed proteases (caspases), that catalyze the relatively orderly biochemical cascade of terminal events known as apoptosis. The present review discusses the evidence that these steps occur after treatment with etoposide or camptothecin, the two prototypic topoisomerase poisons that are commonly studied. As in any emerging area, a large number of questions remain to be answered about the process of cell death induced by topoisomerase-directed drugs.

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Exposure to camptothecin breaks leading and lagging strand simian virus 40 DNA replication forks

Abstract

J. Biol. Chem

Cell

J. Mol. Biol

Mol. Biol

Cell

Science

Nucl. Acid Res

Mol. Pharmacol

Cancer Res