Residues within the N-terminal domain of human topoisomerase I play a direct role in relaxation.
All eukaryotic forms of DNA topoisomerase I contain an extensive and highly charged N-terminal domain. This domain contains several nuclear localization sequences and is essential for in vivo function of the enzyme. However, so far no direct function of the N-terminal domain in the in vitro topoisomerase I reaction has been reported. In this study we have compared the in vitro activities of a truncated form of human topoisomerase I lacking amino acids 1-206 (p67) with the full-length enzyme (p91). Using these enzyme forms, we have identified for the first time a direct role of residues within the N-terminal domain in modulating topoisomerase I catalysis, as revealed by significant differences between p67 and p91 in DNA binding, cleavage, strand rotation, and ligation. A comparison with previously published studies showing no effect of deleting the first 174 or 190 amino acids of topoisomerase I (Stewart, L., Ireton, G. C., and Champoux, J. J. (1999) J. Biol. Chem. 274, 32950-32960; Bronstein, I. B., Wynne-Jones, A., Sukhanova, A., Fleury, F., Ianoul, A., Holden, J. A., Alix, A. J., Dodson, G. G., Jardillier, J. C., Nabiev, I., and Wilkinson, A. J. (1999) Anticancer Res. 19, 317-327) suggests a pivotal role of amino acids 191-206 in catalysis. Taken together the presented data indicate that at least part(s) of the N-terminal domain regulate(s) enzyme/DNA dynamics during relaxation most probably by controlling non-covalent DNA binding downstream of the cleavage site either directly or by coordinating DNA contacts by other parts of the enzyme.
Department of Molecular and Structural Biology, University of Aarhus, C.F. Møllers Allé, Building 130, DK-8000, Aarhus C, Denmark.
M
M
Lisby
J R
JR
Olesen
C
C
Skouboe
B O
BO
Krogh
T
T
Straub
F
F
Boege
S
S
Velmurugan
P M
PM
Martensen
A H
AH
Andersen
M
M
Jayaram
O
O
Westergaard
B R
BR
Knudsen
D002384:Catalysis; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D011487:Protein Conformation
D004264:DNA Topoisomerases, Type I