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PMID: 10805740 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Conversion of topoisomerase I cleavage complexes on the leading strand of ribosomal DNA into 5'-phosphorylated DNA double-strand breaks by replication runoff.

Molecular and cellular biology ·Vol. 20 ·No. 11 ·2000-06-00 ·Pages 3977-87

Strumberg D, Pilon AA, Smith M, Hickey R, Malkas L, Pommier Y

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.

MeSH Terms
Camptothecin/pharmacology DNA/biosynthesis DNA Damage DNA Replication DNA Topoisomerases, Type I/metabolism DNA, Ribosomal/metabolism DNA, Single-Stranded Enzyme Inhibitors/pharmacology HT29 Cells Humans Kinetics Multigene Family Phosphorylation Topoisomerase I Inhibitors
Chemicals
DNA, Ribosomal DNA, Single-Stranded Enzyme Inhibitors Topoisomerase I Inhibitors DNA DNA Topoisomerases, Type I Camptothecin
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Strumberg D
Laboratory of Molecular Pharmacology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4255, USA.
Pilon A A
Smith M
Hickey R
Malkas L
Pommier Y
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.20.11.3977-3987.2000
Published
2000-06-00
Pages
3977-87
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC85758
Subset
IM
External Links
PubMed source DOI: 10.1128/MCB.20.11.3977-3987.2000 PMC Full Text View journal details
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