TY - JOUR

T1 - The Transducer Domain Is Important for Clamp Operation in Human DNA Topoisomerase IIα

AU - Oestergaard, Vibe H.

AU - Bjergbaek, Lotte

AU - Skouboe, Camilla

AU - Giangiacomo, Laura

AU - Knudsen, Birgitta R.

AU - Andersen, Anni H.

PY - 2004/1/16

Y1 - 2004/1/16

N2 - 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 IIα 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.

AB - 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 IIα 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.

UR - http://www.scopus.com/inward/record.url?scp=0347087506&partnerID=8YFLogxK

U2 - 10.1074/jbc.M309624200

DO - 10.1074/jbc.M309624200

M3 - Journal article

C2 - 14583603

AN - SCOPUS:0347087506

VL - 279

SP - 1684

EP - 1691

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 3

ER -