Communication between the ATPase and cleavage/religation domains of human topoisomerase IIalpha.
The DNA strand passage activity of eukaryotic topoisomerase II relies on a cascade of conformational changes triggered by ATP binding to the N-terminal domain of the enzyme. To investigate the interdomain communication between the ATPase and cleavage/religation domains of human topoisomerase IIalpha, we characterized a mutant enzyme that contains a deletion at the interface between the two domains, covering amino acids 350-407. The ATPase domain retained full activity with a rate of ATP hydrolysis that was severalfold higher than normal, but the ATPase activity was unaffected by DNA. The cleavage and religation activities of the enzyme were comparable with those of the wild-type enzyme both in the absence and presence of cancer chemotherapeutic agents. However, neither ATP nor a nonhydrolyzable ATP analog stimulated cleavage complex formation. Although both conserved domains retained full activity, the mutant enzyme was unable to coordinate these activities into strand passage. Our findings suggest that the normal conformational transitions occurring in the enzyme upon ATP binding are hampered or lacking in the mutant enzyme. Consistent with this hypothesis, the enzyme displayed an abnormal clamp closing activity. In summary, the region covering amino acids 350-407 in human topoisomerase IIalpha seems to be essential for correct interdomain communication and probably is involved in signaling ATP binding to the rest of the enzyme.
Bjergbaek
L
L
Department of Molecular and Structural Biology, University of Aarhus, C. F. Mollers Allé, Building 130, 8000 Arhus C, Denmark.
Kingma
P
P
Nielsen
I S
IS
Wang
Y
Y
Westergaard
O
O
Osheroff
N
N
Andersen
A H
AH
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000951:Antigens, Neoplasm; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism; D004268:DNA-Binding Proteins; D017353:Gene Deletion; D005816:Genetic Complementation Test; D006801:Humans; D006868:Hydrolysis; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D010957:Plasmids; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013997:Time Factors
D000951:Antigens, Neoplasm; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D007527:Isoenzymes; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases; D004250:DNA Topoisomerases, Type II