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. 1998 Jun 15;26(12):2963–2970. doi: 10.1093/nar/26.12.2963

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

F Rossi 1, E Labourier 1, I E Gallouzi 1, J Derancourt 1, E Allemand 1, G Divita 1, J Tazi 1
PMCID: PMC147659  PMID: 9611242

Abstract

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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Selected References

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