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Molecular and Cellular Biology, June 2000, p. 3977-3987, Vol. 20, No. 11
Laboratory of Molecular Pharmacology,
Division of Basic Sciences, National Cancer Institute, National
Institutes of Health, Bethesda, Maryland
20892-4255,1 and Department of
Pharmacology and Experimental Therapeutics, University of Maryland,
Baltimore, Maryland 212012
Received 16 November 1999/Returned for modification 5 January
2000/Accepted 3 March 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 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.
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Conversion of Topoisomerase I Cleavage Complexes on
the Leading Strand of Ribosomal DNA into 5'-Phosphorylated DNA
Double-Strand Breaks by Replication Runoff

*
Corresponding author. Mailing address: Laboratory of
Molecular Pharmacology, Bldg. 37, Rm. 5D02, NIH, Bethesda, MD
20892-4255. Phone: (301) 496-5944. Fax: (301) 402-0752. E-mail:
pommier{at}nih.gov.
Present address: Department of Internal Medicine (Cancer Research),
West German Cancer Center, University Medical School, 45122 Essen, Germany.
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