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 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.
West
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Department of Biochemistry and Genetics, The Medical School, The University of Newcastle Upon Tyne, Newcastle Upon Tyne NE2 4HH, UK.
Meczes
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Thorn
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Turnbull
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Marshall
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Austin
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D002384:Catalysis; D002413:Cations, Divalent / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004278:DNA, Superhelical / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D005816:Genetic Complementation Test; D018698:Glutamic Acid / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D008239:Lysine / Q000235:genetics; D008274:Magnesium / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics
D002413:Cations, Divalent; D004271:DNA, Fungal; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D004247:DNA; D004250:DNA Topoisomerases, Type II; D008274:Magnesium; D008239:Lysine