Loss of drug-stimulated topoisomerase II DNA breaks in living cells is different at two unrelated loci.
Topoisomerase II (top2) has been implicated in the initial steps of chromosomal translocations leading to leukemias and lymphomas, since it can generate DNA cleavage. To evaluate the effects of chromatin structure on enzyme-mediated cleavage, we determined the kinetics of loss of double-stranded DNA breaks stimulated by top2 poisons in Drosophila melanogaster Kc cells at two genomic regions that differ in chromatin structure. Moreover, cleavage loss was determined at 25 degrees C as well as after heat shock. Kinetics were dependent on the poison, nevertheless, loss rate overall was slow at the histone gene cluster, an active chromatin domain. At the repressed satellite III DNA, loss of cleavage was much faster and complete after 5 min in drug-free medium. In addition, differences were noted among sites that were closely spaced and equally intense. Following heat shock at 37 degrees C, we observed reduced cleavage levels and faster loss of breaks at the histone gene cluster. In vitro reversal could only partially explain the in vivo kinetics. Thus, the chromatin context of DNA breaks might play a role in the loss of top2 DNA breaks. The present findings suggest that irreversible cuts may more likely occur in active than silent loci.
Binaschi
M
M
Department of Experimental Oncology, Istituto Nazionale Tumori, 20133 Milan, Italy.
Borgnetto
M E
ME
Capranico
G
G
D000818:Animals; D002460:Cell Line; D002843:Chromatin / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004276:DNA, Satellite / Q000235:genetics; D004331:Drosophila melanogaster / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006657:Histones / Q000378:metabolism; D007700:Kinetics; D013696:Temperature; D059005:Topoisomerase II Inhibitors
D002843:Chromatin; D004276:DNA, Satellite; D004791:Enzyme Inhibitors; D006657:Histones; D059005:Topoisomerase II Inhibitors; D004250:DNA Topoisomerases, Type II