Residues 190-210 of human topoisomerase I are required for enzyme activity in vivo but not in vitro.
DNA-topoisomerase I (topo I) unwinds the DNA- double helix by cutting one strand and allowing rotation of the other. In vitro, this function does not require the N-terminal domain of the enzyme, which is believed to regulate cellular properties. To assess this role, we studied the cellular distribution and mobility of green fluorescent protein-chimera of human topo I lacking either the entire N-terminal domain or a portion of it. We find that topo I truncated up to position 210 is not stabilized by camptothecin in covalent DNA-complexes inside a living cell, whereas in vitro it retains full DNA-relaxation activity, and is targeted by camptothecin in the usual manner. This difference is not shared with a fragment lacking the N-terminal domain up to position 190, indicating that residues 190-210 play a crucial role for the activity of the enzyme in its physiological environment, but not in vitro. Since it is impossible to discriminate in vivo whether this region is required for topo I to form covalent DNA intermediates in the cell, or just for camptothecin to bind and stabilize such complexes, we could not explain precisely these cellular observations. However, inactivity in vivo of the enzyme lacking this region is indicated by a lesser cytotoxicity.
Christensen
Morten O
MO
Institute of Clinical Chemistry and Laboratory Diagnostics, Heinrich-Heine-University, Medical School, Moorenstrasse 5, D-40225 Duesseldorf, Germany.
Barthelmes
Hans U
HU
Boege
Fritz
F
Mielke
Christian
C
D002166:Camptothecin / Q000494:pharmacology; D020134:Catalytic Domain; D002460:Cell Line; D002474:Cell-Free System; D004247:DNA / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004351:Drug Resistance / Q000235:genetics; D006801:Humans; D009994:Osmolar Concentration; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012492:Salts / Q000494:pharmacology; D017384:Sequence Deletion / Q000235:genetics*; D059004:Topoisomerase I Inhibitors
D012492:Salts; D059004:Topoisomerase I Inhibitors; D004247:DNA; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin