Conversion of topoisomerase 1 cleavage complexes on the leading strand of ribosomal DNA into 5 '-phosphorylated DNA double-strand breaks by replication runoff
D. Strumberg et al., Conversion of topoisomerase 1 cleavage complexes on the leading strand of ribosomal DNA into 5 '-phosphorylated DNA double-strand breaks by replication runoff, MOL CELL B, 20(11), 2000, pp. 3977-3987
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.