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

• Published in: Nucleic acids research 1998-06, Vol.26 (12), p.2963-2970
• Main Authors: Rossi, Ferdinand, Labourier, Emmanuel, Gallouzi, Imed-eddine, Derancourt, Jean, Allemand, Eric, Divita, Gilles, Tazi, Jamal
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - analogs & derivatives; Adenosine Triphosphate - chemistry; Amino Acid Sequence; Azides - chemistry; Binding Sites; Biochemistry, Molecular Biology; Conserved Sequence; Cross-Linking Reagents; DNA Topoisomerases, Type I - chemistry; DNA Topoisomerases, Type I - genetics; Humans; Kinetics; Life Sciences; Peptide Fragments - analysis; Photoaffinity Labels; Protein Kinases - chemistry; Protein Kinases - genetics; Recombinant Fusion Proteins; Sequence Analysis; Sequence Deletion; Tyrosine - chemistry
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/26.12.2963

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 [α-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.