Hindering the strand passage reaction of human topoisomerase IIalpha without disturbing DNA cleavage, ATP hydrolysis, or the operation of the N-terminal clamp.
DNA topoisomerase II is an essential enzyme that releases a topological strain in DNA by introduction of transient breaks in one DNA helix through which another helix is passed. While changing DNA topology, ATP is required to drive the enzyme through a series of conformational changes dependent on interdomain communication. We have characterized a human topoisomerase IIalpha enzyme with a two-amino acid insertion at position 351 in the transducer domain. The mutation specifically abolishes the DNA strand passage event of the enzyme, probably because of a sterical hindrance of T-segment transport. Thus, the enzyme fails to decatenate and relax DNA, even though it is fully capable of ATP hydrolysis, closure of the N-terminal clamp, and DNA cleavage. The cleavage activity is increased, suggesting that the transducer domain has a role in regulating DNA cleavage. Furthermore, the enzyme has retained a tendency to increase DNA cleavage upon nucleotide binding and also responds to DNA with elevated ATP hydrolysis. However, the DNA-mediated increase in ATP hydrolysis is lower than that obtained with the wild-type enzyme but similar to that of a cleavage-deficient topoisomerase IIalpha enzyme. Our results strongly suggest that the strand passage event is required for efficient DNA stimulation of topoisomerase II-mediated ATP hydrolysis, whereas the stimulation occurs independent of the DNA cleavage reaction per se. A comparison of the strand passage deficient-enzyme described here and the cleavage-deficient enzyme may have applications in other studies where a clear distinction between strand passage and topoisomerase II-mediated DNA cleavage is desirable.
Oestergaard
Vibe H
VH
Department of Molecular Biology, University of Aarhus, C. F. Møllers Allé, Building 130, 8000 Aarhus C, Denmark.
Giangiacomo
Laura
L
Bjergbaek
Lotte
L
Knudsen
Birgitta R
BR
Andersen
Anni H
AH
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D000951:Antigens, Neoplasm; D002499:Centrifugation, Density Gradient; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004270:DNA, Circular / Q000737:chemistry; D004268:DNA-Binding Proteins; D017353:Gene Deletion; D005816:Genetic Complementation Test; D005821:Genetic Techniques; D006801:Humans; D006868:Hydrolysis; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013997:Time Factors
D000951:Antigens, Neoplasm; D004270:DNA, Circular; D004268:DNA-Binding Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D004250:DNA Topoisomerases, Type II