Conversion of topoisomerase 1 cleavage complexes on the leading strand of ribosomal DNA into 5 '-phosphorylated DNA double-strand breaks by replication runoff

Topoisomerase I cleavage complexes can be induced by a variety of DNA damag es and by the anticancer drug camptothecin. We have developed a ligation-me diated PCR (LM-PGR) assay to analyze replication-mediated DNA double-strand breaks induced by topoisomerase I cleavage complexes in human colon carcin oma HT29 cells at the nucleotide level. We found that conversion of topoiso merase I cleavage complexes into replication-mediated DNA double-strand bre aks was only detectable on the leading strand for DNA synthesis, which sugg ests an asymmetry in the way that topoisomerase I cleavage complexes are me tabolized on the two arms of a replication fork. Extension by Tag DNA polym erase was not required for ligation to the LM-PCR primer, indicating that t he 3' DNA ends are extended by DNA polymerase in vivo closely to the 5' end s of the topoisomerase I cleavage complexes. These findings suggest that th e 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, whic h suggests that a DNA 5' kinase activity acts on the double-strand ends gen erated by replication runoff. The replication-mediated DNA double-strand br eaks: were rapidly reversible after cessation of the topoisomerase I cleava ge complexes, suggesting the existence of efficient repair pathways for rem oval of topoisomerase I-DNA covalent adducts in ribosomal DNA.