Mutagenesis of E477 or K505 in the B ' domain of human topoisomerase II beta increases the requirement for magnesium ions during strand passage

A type II topoisomerase is essential for decatenating DNA replication produ cts, and it accomplishes this task by passing one DNA duplex through a tran sient break in a second duplex. The B' domain of topoisomerase II contains three highly conserved motifs, EGDSA, PL(R/K)GK(I/L/M)LNVR, and IMTD(Q/A)DX D. We have investigated these motifs in topoisomerase II beta by mutagenesi s, and report that they play a critical role in establishing the DNA cleava ge-religation equilibrium. In addition, the mutations E477Q (EGDSA) and K50 5E (PLRGKILNVR) increase the optimal magnesium ion concentration for strand passage, without affecting the Mg2+ dependence of ATP hydrolysis. It is li kely that the binding affinity of the magnesium ion(s) specifically require d for DNA cleavage has been reduced by these mutations. The crystal structu re of yeast topo II indicates that residues E477 and K505 may help to posit ion the three aspartate residues of the IMTD(Q/A)DXD motif for magnesium io n coordination, and we propose two possible locations for the magnesium ion binding site(s). These observations are consistent with a previous model i n which the B' domain is positioned such that these acidic residues lie nex t to the active site tyrosine residue. A magnesium ion bound by these aspar tate residues could therefore mediate the DNA cleavage-religation reaction.