A human topoisomerase II alpha heterodimer with only one ATP binding site can go through successive catalytic cycles.
Eukaryotic DNA topoisomerase II is a dimeric nuclear enzyme essential for DNA metabolism and chromosome dynamics. It changes the topology of DNA by coupling binding and hydrolysis of two ATP molecules to the transport of one DNA duplex through a temporary break introduced in another. During this process the structurally and functionally complex enzyme passes through a cascade of conformational changes, which requires intra- and intersubunit communication. To study the importance of ATP binding and hydrolysis in relation to DNA strand transfer, we have purified and characterized a human topoisomerase II alpha heterodimer with only one ATP binding site. The heterodimer was able to relax supercoiled DNA, although less efficiently than the wild type enzyme. It furthermore possessed a functional N-terminal clamp and was sensitive to ICRF-187. This demonstrates that human topoisomerase II alpha can pass through all the conformations required for DNA strand passage and enzyme resetting with binding and hydrolysis of only one ATP. However, the heterodimer lacked the normal stimulatory effect of DNA on ATP binding and hydrolysis as well as the stimulatory effect of ATP on DNA cleavage. The results can be explained in a model, where efficient catalysis requires an extensive communication between the second ATP and the DNA segment to be cleaved.
Skouboe
Camilla
C
Department of Molecular Biology, University of Aarhus, C. F. Møllers Allé, Building 130, Arhus C 8000, Denmark.
Bjergbaek
Lotte
L
Oestergaard
Vibe H
VH
Larsen
Morten K
MK
Knudsen
Birgitta R
BR
Andersen
Anni H
AH
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000951:Antigens, Neoplasm; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D017931:DNA Primers; D004250:DNA Topoisomerases, Type II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins; D019281:Dimerization; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics
D000951:Antigens, Neoplasm; D017931:DNA Primers; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D000266:Adenylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D004250:DNA Topoisomerases, Type II