The C-terminal domain but not the tyrosine 723 of human DNA topoisomerase I active site contributes to kinase activity

Rossi, F.; Labourier, E.; Gallouzi, I. E.; Derancourt, J.; Allemand, E.; Divita, G.; Tazi, J.

Nucleic Acids Res

1998-06-15 / vol 26 / pages 2963-70


Human DNA topoisomerase I not only has DNA relaxing activity, but also splicing factors phosphorylating activity. Topo I shows strong preference for ATP as the phosphate donor. We used photoaffinity labeling with the ATP analogue [alpha-32P] 8-azidoadenosine-5′-triphosphate combined with limited proteolysis to characterize Topo I domains involved in ATP binding. The majority of incorporated analogue was associated with two fragments derived from N-terminal and C-terminal regions of Topo I, respectively. However, mutational analysis showed that deletion of the first 138 N-terminal residues, known to be dispensable for topoisomerase activity, did not change the binding of ATP or the kinase activity. In contrast, deletion of 162 residues from the C-terminal domain was deleterious for ATP binding, kinase and topoisomerase activities. Furthermore, a C-terminal tyrosine 723 mutant lacking topoisomerase activity is still able to bind ATP and to phosphorylate SF2/ASF, suggesting that the two functions of Topo I can be separated. These findings argue in favor of the fact that Topo I is a complex enzyme with a number of potential intra-cellular functions.

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Humans; Amino Acid Sequence; Binding Sites; Kinetics; Sequence Deletion; Adenosine Triphosphate/analogs & derivatives/*chemistry; Azides/chemistry; Conserved Sequence; Cross-Linking Reagents; DNA Topoisomerases, Type I/*chemistry/genetics; Peptide Fragments/analysis; Photoaffinity Labels; Protein Kinases/*chemistry/genetics; Recombinant Fusion Proteins; Sequence Analysis; Tyrosine/chemistry

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