The DNA Cleavage Reaction of Human Type II Topoisomerases

Abstract

The work presented in this dissertation examines the DNA cleavage activity of human topoisomerase II. Type II topoisomerases are required for removing DNA knots and tangles and are important chemotherapy drug targets. The ability to cleave DNA is critical to the cellular and pharmacological functions of human type II topoisomerases. To study the mechanism of DNA cleavage, we developed a system that isolates topoisomerase II-mediated DNA scission from ligation using substrates containing a 3’-bridging phosphorothiolate at the scissile bond. The characteristics of topoisomerase IIá-mediated cleavage of phosphorothiolate oligonucleotides were identical to those seen with wild-type substrates, except that no ligation was observed. The unidirectional accumulation of cleavage complexes enabled us to examine the effects of topoisomerase II poisons on the rate of DNA cleavage and will be valuable for future studies on DNA scission and the topoisomerase II-DNA cleavage complex. Topoisomerase II modulates DNA topology by generating double-stranded breaks in DNA. However, it is unclear how the enzyme coordinates its protomer halves. Results demonstrate that a nick at one scissile bond dramatically increases the rate of cleavage by human topoisomerase IIá at the opposite-strand scissile bond. We propose that this enhanced “second strand” activity coordinates the two topoisomerase II active sites, allowing the enzyme to create double-stranded breaks. We also found that nicks poison topoisomerase II and generate new cleavage sites. Finally, the role of metal ions in the DNA cleavage reactions of human topoisomerase IIá and IIâ were characterized. Results demonstrate that both enzymes employ a two-metal-ion mechanism for DNA cleavage. Also, an interaction between one divalent metal ion and the 3’-bridging atom of the scissile phosphate greatly enhances enzyme-mediated DNA cleavage, most likely by stabilizing the leaving 3’-oxygen. Further, there is an important interaction between a second metal ion and a non-bridging atom of the scissile phosphate that stimulates DNA cleavage mediated by topoisomerase IIâ. Although this non-bridging interaction frequently is postulated in models for enzyme-mediated DNA cleavage, our evidence with topoisomerase IIâ is the first biochemical demonstration that a metal ion contacts this position during scission.