A type II topoisomerase is essential for decatenating DNA replication products, and it accomplishes this task by passing one DNA duplex through a transient 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)DXD. We have investigated these motifs in topoisomerase II beta by mutagenesis, and report that they play a critical role in establishing the DNA cleavage-religation equilibrium. In addition, the mutations E477Q (EGDSA) and K505E (PLRGKILNVR) increase the optimal magnesium ion concentration for strand passage, without affecting the Mg(2+) dependence of ATP hydrolysis. It is likely that the binding affinity of the magnesium ion(s) specifically required for DNA cleavage has been reduced by these mutations. The crystal structure of yeast topo II indicates that residues E477 and K505 may help to position the three aspartate residues of the IMTD(Q/A)DXD motif for magnesium ion coordination, and we propose two possible locations for the magnesium ion binding site(s). These observations are consistent with a previous model in which the B' domain is positioned such that these acidic residues lie next to the active site tyrosine residue. A magnesium ion bound by these aspartate residues could therefore mediate the DNA cleavage-religation reaction.

Topoisomerase II is an essential enzyme which manipulates the topology of DNA. Its activity is important to relieve the DNA supercoiling generated by transcription and replication, and it is required for the condensation and segregation of chromosomes during mitosis and meiosis [l]. Topoisomerase II functions by passing one DNA helix through a transient break in a second DNA helix, a process which is driven by ATP binding and hydrolysis. Several anticancer drugs, for example etoposide and doxorubicin, exert their cytotoxic effects by targeting topoisomerase II. These drugs stabilise the cleaved DNA intermediate in the topoisomerase 11 reaction cycle, and cellular processes convert these ternary complexes into double stranded DNA breaks. There are two human topoisomerase II isofom, a and p, but it is not clear whether the isofom have different roles within the cell, nor whether they are differentially targeted by anticancer drugs.Each topoisomerase II monomer is composed of three well conserved domains and a nonconserved C-terminal tail ( Figure 1). The domains are separated by the protease sensitive regions, A, B and C [2, 31. ATP binding and hydrolysis is carried out by the N-terminal domain, and the active site tyrosine residue, which carries out the DNA cleavage-religation reaction, is located in domain BC. The crystal structure of the central A-C fragment of yeast topoisomerase II has been solved, and this has allowed a refined model for the catalytic cycle of topoisomerase II to be proposed [4].Topoisomerase II has several highly conserved amino acid motifs, including the EGDSA, PL(IUK)GK(VUM)LN and IM(T/A)D(Q/A)DXD motifs which are hydrogen bonded with each other in the A-B domain (Figure 1). The roles of these motifs are UnknOWII, although some mutations conferring drug resistance are located in these regions [5, 61.Figure 1. Domain structure of eukaryotic topoisomerase IIP 50 1-509: PL(RK)GK(YUM)LNV 477-481: EGDSA I 554-561: IM(T/A)D(Q/A)DXD ATPasedomain A B c unconserved Active tail T Y rIn this study, 7 conserved amino acids in these three motifs of human topoisomerase IIP were mutated in order to determine their roles in topoisomerase II activity. The topoisomerase IIP mutants were assayed for their ability to complement temperature sensitive yeast topoisomerase II in vivo. PS480A and PR51OQ were able to weakly complement rs yeast topoisomerase II, whereas the other mutants, PW77Q. PR503E. PK505E. PD557Q and pD561Q could not.The mutant proteins were then overexpressed in yeast and purified to allow detailed characterisation of any alterations to the catalytic cycle. PD557Q and PA561Q were unstable, and so were not analysed further. The other 5 mutants retained between 0.2% and 68% of wild type topoisomerase IIP activity, as assayed by relaxation of supercoiled DNA and decatenation of kinetoplast DNA. ATP hydrolysis activity was assayed by coupling it to NADH oxidation which was measured spectrophotometrically. All the mutants retained greater than 47% of the wild type. topoisomeme I1 ATPase activity.A ...