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J. Biol. Chem., Vol. 283, Issue 24, 16711-16722, June 13, 2008

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Mitotic Phosphorylation Stimulates DNA Relaxation Activity of Human Topoisomerase I^*

Jennifer S. Hackbarth, Marina Galvez-Peralta, Nga T. Dai, David A. Loegering, Kevin L. Peterson, Xue W. Meng, Larry M. Karnitz, and Scott H. Kaufmann¹

From the Department of Biochemistry and Molecular Biology and Division of Oncology Research, Mayo Clinic, Mayo Graduate School, Rochester, Minnesota 55905

Human DNA topoisomerase I (topo I) is an essential mammalian enzyme that regulates DNA supercoiling during transcription and replication. In addition, topo I is specifically targeted by the anticancer compound camptothecin and its derivatives. Previous studies have indicated that topo I is a phosphoprotein and that phosphorylation stimulates its DNA relaxation activity. The locations of most topo I phosphorylation sites have not been identified, preventing a more detailed examination of this modification. To address this issue, mass spectrometry was used to identify four topo I residues that are phosphorylated in intact cells: Ser¹⁰, Ser²¹, Ser¹¹², and Ser³⁹⁴. Immunoblotting using anti-phosphoepitope antibodies demonstrated that these sites are phosphorylated during mitosis. In vitro kinase assays demonstrated that Ser¹⁰ can be phosphorylated by casein kinase II, Ser²¹ can be phosphorylated by protein kinase C, and Ser¹¹² and Ser³⁹⁴ can be phosphorylated by Cdk1. When wild type topo I was pulled down from mitotic cells and dephosphorylated with alkaline phosphatase, topo I activity decreased 2-fold. Likewise, topo I polypeptide with all four phosphorylation sites mutated to alanine exhibited 2-fold lower DNA relaxation activity than wild type topo I after isolation from mitotic cells. Further mutational analysis demonstrated that Ser²¹ phosphorylation was responsible for this change. Consistent with these results, wild type topo I (but not S21A topo I) exhibited increased sensitivity to camptothecin-induced trapping on DNA during mitosis. Collectively these results indicate that topo I is phosphorylated during mitosis at multiple sites, one of which enhances DNA relaxation activity in vitro and interaction with DNA in cells.

Received for publication, September 14, 2007 , and in revised form, March 20, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant R01 CA73709. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4.

¹ To whom correspondence should be addressed: Division of Oncology Research, Guggenheim 1342C, Mayo Clinic, 200 First St. S.W., Rochester, MN 55905. Tel.: 507-284-8950; Fax: 507-284-3906; E-mail: Kaufmann.scott{at}mayo.edu.

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