Trapping of mammalian topoisomerase I and recombinations induced by damaged DNA containing nicks or gaps. Importance of DNA end phosphorylation and camptothecin effects.
We used purified mammalian topoisomerases I (top1) and oligonucleotides containing a unique top1 cleavage site to study top1-mediated cleavage and recombination in the presence of nicks and short gaps mimicking DNA damage. In general, top1 cleavage was not induced opposite to the nicks, and nicks upstream from the top1 cleavage site suppressed top1 activity. Irreversible top1 cleavage complexes ("suicide products" or "aborted complexes") were produced in DNA containing nicks or short gaps just opposite to the normal top1 cleavage site. Camptothecin enhanced the formation of the aborted top1 complexes only for nicks downstream from the cleavage site. These aborted (suicide) complexes can mediate DNA recombination and promote illegitimate recombination by catalyzing the ligation of nonhomologous DNA fragments (acceptors). We report for the first time that top1-mediated recombination is greatly enhanced by the presence of a phosphate at the 5' terminus of the top1 aborted complex (donor DNA). By contrast, phosphorylation of the 3' terminus of the gap did not affect recombination. At concentrations that strongly enhanced inhibition of intramolecular religation, resulting in an increase of top1 cleavable complexes, camptothecin did not reduce recombination (intermolecular religation). Nicks or gaps with 5'-phosphate termini would be expected to be produced directly by ionizing radiations or by processing of abasic sites and DNA lesions induced by carcinogens or drugs used in cancer chemotherapy. Thus, these results further demonstrate that DNA damage can efficiently trap top1-cleavable complexes and enhance top1-mediated DNA recombination.
Pourquier
P
P
Laboratory of Molecular Pharmacology, Division of Basic Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892-4255, USA.
Pilon
A A
AA
Kohlhagen
G
G
Mazumder
A
A
Sharma
A
A
Pommier
Y
Y
D002166:Camptothecin / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D010766:Phosphorylation; D011995:Recombination, Genetic; D059004:Topoisomerase I Inhibitors
D004791:Enzyme Inhibitors; D059004:Topoisomerase I Inhibitors; D004247:DNA; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin