Topoisomerase I poisoning results in PARP-mediated replication fork reversal (original) (raw)
Related papers
Mutation Research Letters, 1993
Previous studies have shown the importance of DNA replication fork progression for the cytotoxicity of topoisomerase inhibitors. Nevertheless, while it was concluded that an interference of moving forks with drug-stabilized topo I-DNA complexes is critical for cell death, in the case of topo II only a partial contribution to cell killing was proposed. We have studied the influence of inhibition of DNA replication by aphidicolin on the production of chromosomal aberrations and SCE by topoisomerase inhibitors. Our results seem to indicate that fork progression is necessary for both cytogenetic endpoints. Pulsed field gel electrophoresis also confirmed this conclusion at the level of DNA breakage (double-strand breaks) efficiently induced by m-AMSA treatment alone, but not when aphidicolin was present. Differences found between topo I and topo II inhibitors (camptothecin and m-AMSA, respectively) could be explained as due to differences, in their persistence in trapping the 'cleavable complex'.
Human RECQ1 promotes restart of replication forks reversed by DNA topoisomerase I inhibition
Nature Structural & Molecular Biology, 2013
Topoisomerase I (TOP) inhibitors are an important class of anticancer drugs. The cytotoxicity of TOP inhibitors can be modulated by replication fork reversal through a process that requires poly(ADP-ribose) polymerase (PARP) activity. Whether regressed forks can efficiently restart and what factors are required to restart fork progression after fork reversal are still unknown. We have combined biochemical and EM approaches with single-molecule DNA fiber analysis to identify a key role for human RECQ helicase in replication fork restart after TOP inhibition that is not shared by other human RecQ proteins. We show that the poly(ADP-ribosyl)ation activity of PARP stabilizes forks in the regressed state by limiting their restart by RECQ. These studies provide new mechanistic insights into the roles of RECQ and PARP in DNA replication and offer molecular perspectives to potentiate chemotherapeutic regimens based on TOP inhibition.
Cell Reports, 2016
During transcription and DNA replication, the DNA template is overwound ahead of RNA and DNA polymerases and relaxed by DNA topoisomerases. Inhibitors of topoisomerases are potent anticancer agents. Camptothecin traps topoisomerase I on DNA and exerts preferential cytotoxicity toward cancer cells by way of its interference with the progression of replication forks. Starting with an unbiased proteomic analysis, we find that the chromatin remodeling complex BAZ1B-SMARCA5 accumulates near replication forks in camptothecin-exposed cells. We report that BAZ1B associates with topoisomerase I and facilitates its access to replication forks. Single-molecule analyses of replication structures show that BAZ1B contributes to replication interference by camptothecin. A lack of BAZ1B confers increased cellular tolerance of camptothecin. These findings reveal BAZ1B as a key facilitator of topoisomerase I function during DNA replication that affects the response of cancer cells to topoisomerase I inhibitors.
Proceedings of The National Academy of Sciences, 1999
The antitumor agent camptothecin targets DNA topoisomerase I by reversibly stabilizing a covalent enzyme-DNA intermediate. The subsequent collision of DNA replication forks with these drug-enzyme-DNA complexes produces the cytotoxic DNA lesions that signal cell cycle arrest and ultimately lead to cell death. Despite intense investigation, the character of the lesions produced and the repair processes that resolve the damage remain
Biochemistry, 1997
Recent studies have shown that the anticancer drugs VM-26 and mitoxantrone stabilize preferentially the binding of topoisomerase IIR to replicating compared to nonreplicating DNA. To further understand the mechanisms by which cleavable complex-forming topoisomerase II inhibitors interfere with DNA replication, we examined the effects of VM-26 on this process in human leukemia CEM cells. Both the inhibition of DNA synthesis and cell survival were directly related to the total amount of drugstabilized cleavable complexes formed in VM-26-treated cells. DNA chain elongation was also inhibited in a concentration-dependent fashion in these cells, which suggested that VM-26-stabilized cleavable complexes interfered with the movement of DNA replication forks. To test this hypothesis directly, we monitored replication fork progression at a specific site of VM-26-induced DNA cleavage. A topoisomerase II-mediated cleavage site was detected in the first exon of the c-myc gene in VM-26-treated cells. This cleavage site was downstream of a putative replication origin located in the 5′ flanking region of the gene. Replication forks, which moved through this region of the c-myc gene in the 5′ to 3′ direction, were specifically arrested at this site in VM-26-treated cells, but not in untreated or aphidicolin-treated cells. These studies provide the first direct evidence that a VM-26-stabilized topoisomerase II-DNA cleavable complex acts as a replication fork barrier at a specific genomic site in mammalian cells. Furthermore, the data support the hypothesis that the replication fork arrest induced by cleavable complexforming topoisomerase II inhibitors leads to the generation of irreversible DNA damage and cytotoxicity in proliferating cells. fork; SDS, sodium dodecyl sulfate; SSC, sodium chloride-sodium citrate buffer; topo II, DNA topoisomerase II; TCA, trichloroacetic acid; VP-16 (etoposide), 4′demethylepipodophyllotoxin 9-(4,6-O-ethylidene--D-glucopyranoside); VM-26 (teniposide), 4′-demethylepipodopyllotoxin 9-(4,6-O-2-thenylidene--D-glucopyranoside).
Molecular and Cellular Biology, 2000
Topoisomerase I cleavage complexes can be induced by a variety of DNA damages and by the anticancer drug camptothecin. We have developed a ligation-mediated PCR (LM-PCR) assay to analyze replication-mediated DNA double-strand breaks induced by topoisomerase I cleavage complexes in human colon carcinoma HT29 cells at the nucleotide level. We found that conversion of topoisomerase I cleavage complexes into replication-mediated DNA double-strand breaks was only detectable on the leading strand for DNA synthesis, which suggests an asymmetry in the way that topoisomerase I cleavage complexes are metabolized on the two arms of a replication fork. Extension by Taq DNA polymerase was not required for ligation to the LM-PCR primer, indicating that the 3′ DNA ends are extended by DNA polymerase in vivo closely to the 5′ ends of the topoisomerase I cleavage complexes. These findings suggest that the replication-mediated DNA double-strand breaks generated at topoisomerase I cleavage sites are p...
Yeast recombination pathways triggered by topoisomerase II-mediated DNA breaks
Nucleic Acids Research, 2003
Topoisomerase II is a ubiquitous enzyme that removes knots and tangles from the genetic material by generating transient double-strand DNA breaks. While the enzyme cannot perform its essential cellular functions without cleaving DNA, this scission activity is inherently dangerous to chromosomal integrity. In fact, etoposide and other clinically important anticancer drugs kill cells by increasing levels of topoisomerase II-mediated DNA breaks. Cells rely heavily on recombination to repair double-strand DNA breaks, but the speci®c pathways used to repair topoisomerase II-generated DNA damage have not been de®ned. Therefore, Saccharomyces cerevisiae was used as a model system to delineate the recombination pathways that repair DNA breaks generated by topoisomerase II. Yeast cells that expressed wild-type or a drughypersensitive mutant topoisomerase II or overexpressed the wild-type enzyme were examined. Based on cytotoxicity and recombination induced by etoposide in different repair-de®cient genetic backgrounds, double-strand DNA breaks generated by topoisomerase II appear to be repaired primarily by the single-strand invasion pathway of homologous recombination. Non-homologous end joining also was triggered by etoposide treatment, but this pathway was considerably less active than singlestrand invasion and did not contribute signi®cantly to cell survival in S.cerevisiae.
Repair of Topoisomerase I‐Mediated DNA Damage
Progress in Nucleic Acid Research and Molecular Biology, 2006
Topoisomerase I (Top1) is an abundant and essential enzyme. Top1 is the selective target of camptothecins, which are effective anticancer agents. Top1-DNA cleavage complexes can also be trapped by various endogenous and exogenous DNA lesions including mismatches, abasic sites and carcinogenic adducts. Tyrosyl-DNA phosphodiesterase (Tdp1) is one of the repair enzymes for Top1-DNA covalent complexes. Tdp1 forms a multiprotein complex that includes poly(ADP) ribose polymerase (PARP). PARP-deficient cells are hypersensitive to camptothecins and functionally deficient for Tdp1. We will review recent developments in several pathways involved in the repair of Top1 cleavage complexes and the role of Chk1 and Chk2 checkpoint kinases in the cellular responses to Top1 inhibitors. The genes conferring camptothecin hypersensitivity are compiled for humans, budding yeast and fission yeast.