title: Conversion of topoisomerase I cleavage complexes on the leading strand of ribosomal DNA into 5'-phosphorylated DNA double-strand breaks by replication runoff.
abstract: 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 produced by replication runoff. We also found that the 5' ends of these DNA double-strand breaks are phosphorylated in vivo, which suggests that a DNA 5' kinase activity acts on the double-strand ends generated by replication runoff. The replication-mediated DNA double-strand breaks were rapidly reversible after cessation of the topoisomerase I cleavage complexes, suggesting the existence of efficient repair pathways for removal of topoisomerase I-DNA covalent adducts in ribosomal DNA.
authors.affiliation: Laboratory of Molecular Pharmacology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4255, USA.
authors.forename: D
authors.initials: D
authors.lastname: Strumberg
authors.forename: A A
authors.initials: AA
authors.lastname: Pilon
authors.forename: M
authors.initials: M
authors.lastname: Smith
authors.forename: R
authors.initials: R
authors.lastname: Hickey
authors.forename: L
authors.initials: L
authors.lastname: Malkas
authors.forename: Y
authors.initials: Y
authors.lastname: Pommier
mesh_terms: D002166:Camptothecin / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis; D004249:DNA Damage; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004275:DNA, Ribosomal / Q000378:metabolism*; D004277:DNA, Single-Stranded; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019073:HT29 Cells; D006801:Humans; D007700:Kinetics; D005810:Multigene Family; D010766:Phosphorylation; D059004:Topoisomerase I Inhibitors
chemical_list: D004275:DNA, Ribosomal; D004277:DNA, Single-Stranded; D004791:Enzyme Inhibitors; D059004:Topoisomerase I Inhibitors; D004247:DNA; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin