Use of Divalent Metal Ions in the DNA Cleavage Reaction of Human Type II Topoisomerases

Abstract

All type II topoisomerases require divalent metal ions to cleave and ligate DNA. To further elucidate the mechanistic basis for these critical enzyme-mediated events, the role of the metal ion in the DNA cleavage reaction of human topoisomerase IIbeta was characterized and compared to that of topoisomerase IIalpha. This study utilized divalent metal ions with varying thiophilicities in conjunction with DNA cleavage substrates that substituted a sulfur atom for the 3'-bridging oxygen or the nonbridging oxygens of the scissile phosphate. On the basis of time courses of DNA cleavage, cation titrations, and metal ion mixing experiments, we propose the following model for the use of divalent metal ions by human type II topoisomerases. First, both enzymes employ a two-metal ion mechanism to support DNA cleavage. Second, 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. Third, there is an important interaction between a divalent second metal ion and a nonbridging atom of the scissile phosphate that stimulates DNA cleavage mediated by topoisomerase IIbeta. If this interaction exists in topoisomerase IIalpha, its effects on DNA cleavage are equivocal. This last aspect of the model highlights a difference in metal ion utilization during DNA cleavage mediated by human topoisomerase IIalpha and IIbeta.