TY - JOUR
T1 - The C-terminal domain but not the tyrosine 723 of human DNA topoisomerase I active site contributes to kinase activity
AU - Rossi, Ferdinand
AU - Labourier, Emmanuel
AU - Gallouzi, Imed Eddine
AU - Derancourt, Jean
AU - Allemand, Eric
AU - Divita, Gilles
AU - Tazi, Jamal
N1 - Generated from Scopus record by KAUST IRTS on 2022-09-13
PY - 1998/6/15
Y1 - 1998/6/15
N2 - 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.
AB - 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.
UR - https://academic.oup.com/nar/article-lookup/doi/10.1093/nar/26.12.2963
UR - http://www.scopus.com/inward/record.url?scp=0032526626&partnerID=8YFLogxK
U2 - 10.1093/nar/26.12.2963
DO - 10.1093/nar/26.12.2963
M3 - Article
SN - 0305-1048
VL - 26
SP - 2963
EP - 2970
JO - Nucleic Acids Research
JF - Nucleic Acids Research
IS - 12
ER -