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Molecular and Cellular Biology, June 2000, p. 3977-3987, Vol. 20, No. 11
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

Dirk Strumberg,1,dagger André A. Pilon,1 Melanie Smith,2 Robert Hickey,2 Linda Malkas,2 and Yves Pommier1,*

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.

* 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.

dagger Present address: Department of Internal Medicine (Cancer Research), West German Cancer Center, University Medical School, 45122 Essen, Germany.

Molecular and Cellular Biology, June 2000, p. 3977-3987, Vol. 20, No. 11
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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