Residues within the N-terminal Domain of Human Topoisomerase I Play a Direct Role in Relaxation

• Published in: The Journal of biological chemistry Vol. 276; no. 23; pp. 20220 - 20227
• Main Authors: Lisby, Michael, Olesen, Jens R., Skouboe, Camilla, Krogh, Berit O., Straub, Tobias, Boege, Fritz, Velmurugan, Soundarapaudian, Martensen, Pia M., Andersen, Anni H., Jayaram, Makkuni, Westergaard, Ole, Knudsen, Birgitta R.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 08.06.2001; American Society for Biochemistry and Molecular Biology
• Subjects: Catalysis; DNA Topoisomerases, Type I - chemistry; DNA Topoisomerases, Type I - isolation & purification; DNA Topoisomerases, Type I - metabolism; Electrophoresis, Polyacrylamide Gel; Humans; Protein Conformation
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M010991200

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 forin vivo function of the enzyme. However, so far no direct function of the N-terminal domain in the in vitrotopoisomerase 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.