Enhanced processing of UVA-irradiated DNA by human topoisomerase II in living cells.
Solar UV light induces a variety of DNA lesions in the genome. Enhanced cleavage of such base modifications by topoisomerase II has been demonstrated in vitro, but it is unclear what will arise from an interplay of these mechanisms in the genome of a living cell exposed to UV light. To address this question, we have subjected cells expressing biofluorescent topoisomerase IIalpha or IIbeta to DNA base modifications inflicted by a UVA laser at 364 nm through a confocal microscope in a locally confined manner. At DNA sites thus irradiated, we observed rapid, long term (>90 min) accumulation of topoisomerase IIalpha and IIbeta, which was accompanied by a decrease in mobility but not immobilization of the enzyme. The catalytic topoisomerase II inhibitor ICRF-187 prevented the effect when added to the cell culture before the UVA pulse but promoted it when added thereafter. Self-primed in situ extension with rhodamine-dUTP revealed massive DNA breakage at the UVA-exposed spot. Culturing the cells with ICRF-187 before UVA-exposure prevented such breaks. In conclusion, we show in a living cell nucleus that UVA-modified DNA is preferentially targeted and processed by topoisomerase IIalpha and IIbeta. This results in increased levels of topoisomerase II-mediated DNA breaks, but formation of immobile, stable topoisomerase II.DNA intermediates is not notably promoted. Inhibition of topoisomerase II activity by ICRF-187 greatly diminishes UVA-induced DNA breakage, implying topoisomerase IIalpha and IIbeta as endogenous co-factors modulating and possibly aggravating the impact of UVA light on the genome.
Mielke
Christian
C
Institute of Clinical Chemistry and Laboratory Diagnostics, Heinrich-Heine-University, Medical School, Moorenstrasse 5, D-40225 Düsseldorf, Germany. christian.mielke@med.uni-duesseldorf.de
Christensen
Morten O
MO
Barthelmes
Hans Ullrich
HU
Boege
Fritz
F
D000951:Antigens, Neoplasm; D002460:Cell Line; D004247:DNA / Q000378:metabolism / Q000528:radiation effects*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004268:DNA-Binding Proteins; D006801:Humans; D007668:Kidney; D007700:Kinetics; D011929:Razoxane / Q000494:pharmacology; D059005:Topoisomerase II Inhibitors; D014162:Transfection; D014466:Ultraviolet Rays
D000951:Antigens, Neoplasm; D004268:DNA-Binding Proteins; D059005:Topoisomerase II Inhibitors; D011929:Razoxane; D004247:DNA; D004250:DNA Topoisomerases, Type II