title: The C-terminal 20 Amino Acids of Drosophila Topoisomerase 2 Are Required for Binding to a BRCA1 C Terminus (BRCT) Domain-containing Protein, Mus101, and Fidelity of DNA Segregation.
abstract: Eukaryotic topoisomerase 2 (Top2) and one of its interacting partners, topoisomerase IIβ binding protein 1 (TopBP1) are two proteins performing essential cellular functions. We mapped the interacting domains of these two proteins using co-immunoprecipitation and pulldown experiments with truncated or mutant Drosophila Top2 with various Ser-to-Ala substitutions. We discovered that the last 20 amino acids of Top2 represent the key region for binding with Mus101 (the Drosophila homolog of TopBP1) and that phosphorylation of Ser-1428 and Ser-1443 is important for Top2 to interact with the N terminus of Mus101, which contains the BRCT1/2 domains. The interaction between Mus101 and the Top2 C-terminal regulatory domain is phosphorylation-dependent because treatment with phosphatase abolishes their association in pulldown assays. The binding affinity of N-terminal Mus101 with a synthetic phosphorylated peptide spanning the last 25 amino acids of Top2 (with Ser(P)-1428 and Ser(P)-1443) was determined by surface plasmon resonance with a Kd of 0.57 μm In an in vitro decatenation assay, Mus101 can specifically reduce the decatenation activity of Top2, and dephosphorylation of Top2 attenuates this response. Next, we endeavored to establish a cellular system for testing the biological function of Top2-Mus101 interaction. Top2-silenced S2 cells rescued by Top2Δ20, Top2 with 20 amino acids truncated from the C terminus, developed abnormally high chromosome numbers, which implies that Top2-Mus101 interaction is important for maintaining the fidelity of chromosome segregation during mitosis.
authors.affiliation: From the Department of Biochemistry and.
authors.forename: Yu-Tsung Shane
authors.initials: YT
authors.lastname: Chen
authors.affiliation: From the Department of Biochemistry and.
authors.forename: Jianhong
authors.initials: J
authors.lastname: Wu
authors.affiliation: From the Department of Biochemistry and.
authors.forename: Paul
authors.initials: P
authors.lastname: Modrich
authors.affiliation: From the Department of Biochemistry and the Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 115, Taiwan Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710 and hsieh002@gate.sinica.edu.tw.
authors.forename: Tao-Shih
authors.initials: TS
authors.lastname: Hsieh
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D020090:Chromosome Segregation; D059006:Chromosomes, Insect / Q000235:genetics; D004250:DNA Topoisomerases, Type II / Q000737:chemistry* / Q000378:metabolism; D018105:DNA, Kinetoplast / Q000502:physiology; D029721:Drosophila Proteins / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology*; D008969:Molecular Sequence Data; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs
chemical_list: D018797:Cell Cycle Proteins; D018105:DNA, Kinetoplast; D029721:Drosophila Proteins; C416162:mus101 protein, Drosophila; D004250:DNA Topoisomerases, Type II
keywords: DNA replication; DNA topoisomerase; Drosophila; RNA interference (RNAi); chromosome missegregation; protein phosphorylation