The transducer domain is important for clamp operation in human DNA topoisomerase IIalpha.
DNA topoisomerase II is a multidomain homodimeric enzyme that changes DNA topology by coupling ATP hydrolysis to the transport of one DNA helix through a transient double-stranded break in another. The process requires dramatic conformational changes including closure of an ATP-operated clamp, which is comprised of two N-terminal domains from each protomer. The most N-terminal domain contains the ATP-binding site and is directly involved in clamp closure, undergoing dimerization upon ATP binding. The second domain, the transducer domain, forms the walls of the N-terminal clamp and connects the clamp to the enzyme core. Although structurally conserved, it is unclear whether the transducer domain is involved in clamp mechanism. We have purified and characterized a human topoisomerase II alpha enzyme with a two-amino acid insertion at position 408 in the transducer domain. The enzyme retains both ATPase and DNA cleavage activities. However, the insertion, which is situated far from the N-terminal dimerization area, severely disrupts the function of the N-terminal clamp. The clamp-deficient enzyme is catalytically inactive and lacks most aspects of interdomain communication. Surprisingly, it seems to have retained the intersubunit communication, allowing it to bind ATP cooperatively in the presence of DNA. The results show that even distal parts of the transducer domain are important for the dynamics of the N-terminal clamp and furthermore indicate that stable clamp closure is not required for cooperative binding of ATP.
Department of Molecular Biology, University of Aarhus, C. F. Møllers Allé, Building 130, 8000 Aarhus C, Denmark.
Vibe H
VH
Oestergaard
Lotte
L
Bjergbaek
Camilla
C
Skouboe
Laura
L
Giangiacomo
Birgitta R
BR
Knudsen
Anni H
AH
Andersen
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000951:Antigens, Neoplasm; D004247:DNA / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000737:chemistry* / Q000502:physiology; D004268:DNA-Binding Proteins; D006801:Humans; D011487:Protein Conformation
D000951:Antigens, Neoplasm; D004268:DNA-Binding Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases; D004250:DNA Topoisomerases, Type II