Mitotic phosphorylation stimulates DNA relaxation activity of human topoisomerase I.

The Journal of Biological Chemistry
Jennifer S HackbarthScott H Kaufmann

Abstract

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: Ser(10), Ser(21), Ser(112), and Ser(394). Immunoblotting using anti-phosphoepitope antibodies demonstrated that these sites are phosphorylated during mitosis. In vitro kinase assays demonstrated that Ser(10) can be phosphorylated by casein kinase II, Ser(21) can be phosphorylated by protein kinase Calpha, and Ser(112) and Ser(394) 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 DN...Continue Reading

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Apr 18, 2012·PloS One·Yee-Fun SuJaulang Hwang
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