Using 3'-bridging phosphorothiolates to isolate the forward DNA cleavage reaction of human topoisomerase IIalpha.
The ability to cleave DNA is critical to the cellular and pharmacological functions of human type II topoisomerases. However, the low level of cleavage at equilibrium and the tight coupling of the cleavage and ligation reactions make it difficult to characterize the mechanism by which these enzymes cut DNA. Therefore, to establish a system that isolates topoisomerase II-mediated DNA scission from ligation, oligonucleotide substrates were developed that contained a 3'-bridging phosphorothiolate at the scissile bond. Scission of these substrates generates a 3'-terminal -SH moiety that is a poor nucleophile relative to the normal 3'-terminal -OH group. Consequently, topoisomerase II cannot efficiently ligate phosphorothiolate substrates once they are cleaved. The characteristics of topoisomerase IIalpha-mediated cleavage of phosphorothiolate oligonucleotides were identical to those seen with wild-type substrates, except that no ligation was observed. This unidirectional accumulation of cleavage complexes provided critical information regarding coordination of the protomer subunits of topoisomerase IIalpha and the mechanism of action of topoisomerase II poisons. Results indicate that the two enzyme subunits are partially coordinated and that cleavage at one scissile bond increases the degree of cleavage at the other. Furthermore, anticancer drugs such as etoposide and amsacrine that strongly inhibit topoisomerase II-mediated DNA ligation have little effect on the forward scission reaction. In contrast, abasic sites that increase levels of cleavage complexes without affecting ligation stimulate the forward rate of scission. Phosphorothiolate substrates provide significant advantages over traditional "suicide substrates" and should be valuable for future studies on DNA scission and the topoisomerase II-DNA cleavage complex.
Deweese
Joseph E
JE
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Burgin
Alex B
AB
Osheroff
Neil
N
D000951:Antigens, Neoplasm / Q000378:metabolism*; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D017931:DNA Primers; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D009943:Organophosphorus Compounds / Q000737:chemistry*; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D000951:Antigens, Neoplasm; D017931:DNA Primers; D004268:DNA-Binding Proteins; D009943:Organophosphorus Compounds; D013438:Sulfhydryl Compounds; D004247:DNA; D004250:DNA Topoisomerases, Type II