The C-terminal domain but not the tyrosine 723 of human DNA topoisomerase I active site contributes to kinase activity.
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.
Rossi
F
F
Institut de Génétique Moléculaire de Montpellier (IGM), UMR 5535 CNRS, Université Montpellier II, CNRS 1919, route de Mende, F34293 Montpellier Cedex 5, France.
Labourier
E
E
Gallouzi
I E
IE
Derancourt
J
J
Allemand
E
E
Divita
G
G
Tazi
J
J
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry*; D000595:Amino Acid Sequence; D001386:Azides / Q000737:chemistry; D001665:Binding Sites; D017124:Conserved Sequence; D003432:Cross-Linking Reagents; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000032:analysis; D019920:Photoaffinity Labels; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins; D017421:Sequence Analysis; D017384:Sequence Deletion; D014443:Tyrosine / Q000737:chemistry
D001386:Azides; D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D004264:DNA Topoisomerases, Type I