Metal Ion Interactions in the DNA Cleavage/Ligation Active Site of Human Topoisomerase IIα
journal contributionposted on 29.09.2009, 00:00 by Joseph E. Deweese, F. Peter Guengerich, Alex B. Burgin, Neil Osheroff
Human topoisomerase IIα utilizes a two-metal-ion mechanism for DNA cleavage. One of the metal ions (M12+) is believed to make a critical interaction with the 3′-bridging atom of the scissile phosphate, while the other (M22+) is believed to interact with a nonbridging oxygen of the scissile phosphate. Based on structural and mutagenesis studies of prokaryotic nucleic acid enzymes, it has been proposed that the active site divalent metal ions interact with type II topoisomerases through a series of conserved acidic amino acid residues. The homologous residues in human topoisomerase IIα are E461, D541, D543, and D545. To address the validity of these assignments and to delineate interactions between individual amino acids and M12+ and M22+, we individually mutated each of these acidic amino acid residues in topoisomerase IIα to either cysteine or alanine. Mutant enzymes displayed a marked loss of catalytic and DNA cleavage activity as well as a reduced affinity for divalent metal ions. Additional experiments determined the ability of wild-type and mutant topoisomerase IIα enzymes to cleave an oligonucleotide substrate that contained a sulfur atom in place of the 3′-bridging oxygen of the scissile phosphate in the presence of Mg2+, Mn2+, or Ca2+. On the basis of the results of these studies, we conclude that the four acidic amino acid residues interact with metal ions in the DNA cleavage/ligation active site of topoisomerase IIα. Furthermore, we propose that M12+ interacts with E461, D543, and D545 and M22+ interacts with E461 and D541.