Effects of DNA Supercoil Handedness on Catenation by Type II Topoisomerases

Abstract

Type II topoisomerases interact differently with DNA substrates of opposite supercoil handedness: although the genome is globally unwound, DNA ahead of replication or transcription complexes is overwound, and therefore DNA geometry can influence where the enzyme acts during nucleic acid processes. Human topoisomerase IIa and bacterial topoisomerase IV relax positively supercoiled [(+)SC] DNA faster than negatively supercoiled [(-)SC] molecules, while human topoisomerase IIb shows no preference. An important function of topoisomerase IIa and topoisomerase IV is the decatenation of DNA prior to mitosis. Cellular studies in yeast and bacteria indicate that DNA becomes positively supercoiled through interaction with cellular factors prior to decatenation, but it is not yet known whether the enzyme displays an intrinsic preference for decatenating (+)SC DNA or if these additional cellular factors accelerate the reaction. To determine whether type II topoisomerases possess an intrinsic ability to recognize DNA supercoil handedness during catenation and decatenation, we have carried out a series of in vitro experiments. As these two reactions are in equilibrium, it is important to study the effects of DNA supercoil handedness on both of them. Our studies focused on the effects of (-)SC, (+)SC, and relaxed DNA on catenation by type II topoisomerases. Topoisomerase IIa catenated relaxed DNA ~10-fold faster than (-)SC DNA, and ~45-fold faster than (+)SC DNA. The C-terminal domain of topoisomerase IIa has been previously shown to confer the ability of the enzyme to preferentially relax (+)SC DNA over (-)SC DNA. A deletion construct of topoisomerase IIa that lacked the C-terminal domain showed that this enzyme domain is also required for the preferential catenation of relaxed and (-)SC DNA. Catenation assays with (+)SC substrates did not go to completion, suggesting that chromosomes are less likely to be catenated following positive supercoiling. Additional studies performed with topoisomerase IIa indicated that (-)SC DNA does not relax prior to catenation and, at high concentrations, the enzyme can catenate DNA with very low concentrations of ATP as compared to other strand passage reactions. Topoisomerase IIb displayed slower rates of catenation than topoisomerase IIa and did not discriminate between different supercoil handedness states. In contrast, topoisomerase IV preferentially catenated (+)SC DNA over (-)SC and relaxed DNA. Results show that topoisomerase IIa and topoisomerase IV can distinguish supercoil handedness during catenation and may help determine whether (+)SC decatenation uses a different control mechanism.