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Query: EC:5.99.1.2 (topoisomerase)
 9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The decatenation activity of DNA topoisomerase II is essential for viability as eukaryotic cells traverse mitosis. Phosphorylation has been shown to stimulate topoisomerase II activity in vitro. Here we show that topoisomerase II is a phosphoprotein in yeast and that the level of incorporated phosphate is significantly higher at mitosis than in G1. Comparison of tryptic phosphopeptide maps reveals that the major phosphorylation sites in vivo are targets for casein kinase II. Incorporation of phosphate into topoisomerase II is nearly undetectable at the non-permissive temperature in a conditional casein kinase II mutant. The sites modified by casein kinase II are located in the extreme C-terminal domain of topoisomerase II. This domain is absent in prokaryotic and highly divergent among eukaryotic type II topoisomerases, and may serve to regulate functions of topoisomerase II that are unique to eukaryotic cells.
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PMID:Casein kinase II phosphorylates the eukaryote-specific C-terminal domain of topoisomerase II in vivo. 131 74

Type II topoisomerase has been purified from mouse FM3A cells by using P4 phage knotted DNA as a substrate. Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands of apparent molecular masses of 167 and 151 kDa. Partial digestion of the two bands with Staphylococcus aureus V8 protease indicated that the two polypeptides were structurally related. The enzyme required ATP and Mg2+ for activity. dATP could substitute for ATP, and ITP was slightly effective at 5-10 mM. The activity was sensitive to 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), coumermycin, and ethidium bromide. A protein kinase activity was detected in the partially purified topoisomerase II fraction, and this protein kinase was further purified. The protein kinase phosphorylated the purified topoisomerase II, and the phosphorylation of topoisomerase II by the kinase increased the activity by 8.6-fold over that of the unmodified enzyme. The treatment of the purified topoisomerase II with alkaline phosphatase abolished the enzyme activity almost completely, and the treatment of the dephosphorylated topoisomerase II with the protein kinase restored the enzyme activity. The protein kinase activity was not stimulated by Ca2+ or cyclic nucleotides, and the aminoacyl residue phosphorylated by the kinase was serine. Enzymatic properties of the kinase were very similar to those of the kinase reported to be tightly associated with the Drosophila topoisomerase II [Sander, M., Nolan, J. M., & Hsieh, T.-S. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 6938-6942]. The immunoprecipitation of nuclear extracts prepared from 32P-labeled cells with anti-mouse topoisomerase II antiserum indicated that DNA topoisomerase II existed in mouse cells as a phosphoprotein.
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PMID:Purification and characterization of type II DNA topoisomerase from mouse FM3A cells: phosphorylation of topoisomerase II and modification of its activity. 215 52

We have determined that the major mitotic phosphoprotein in chromosomes recognized by the antiphosphoprotein antibody MPM-2 is the 170-kDa isoform of topoisomerase II (topo II), the isoform predominant in proliferating cells. As a prerequisite to making this discovery, it was necessary to develop protocols to protect chromosomal proteins from dephosphorylation during cell extraction and chromosome isolation procedures. Immunofluorescence analysis of the large chromosomes prepared from Indian Muntjac cells revealed colocalization of MPM-2 and anti-topo II antibodies to the chromosomal centromeres and to the axial regions of the chromosomal arms. For biochemical fractionation studies, large quantities of chromosomes from the P388D1 mouse lymphocyte cell line were isolated and treated to remove DNA and histone proteins. Immunoblot and immunoprecipitation experiments with this chromosome scaffold fraction identified the major MPM-2-reactive phosphoprotein to be DNA topo II. Using a panel of anti-peptide antibodies specific to the isoforms of topo II, we determined that the major phosphoprotein recognized by MPM-2 is the 170-kDa isoform of topo II, topo II alpha. The 180-kDa isoform, topo II beta, present in the isolated chromosomes in much smaller quantities, is also recognized by MPM-2. The mitotic phosphorylation of the topo II proteins may be critical for proper chromosome condensation and segregation.
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PMID:DNA topoisomerase II alpha is the major chromosome protein recognized by the mitotic phosphoprotein antibody MPM-2. 769 Sep 61

DNA topoisomerase II is an essential nuclear enzyme required for the proper condensation and segregation of chromosomes during mitotic and meiotic cell division. The enzyme exists in the cell as a phosphoprotein, and it is most highly phosphorylated in G2 and M-phases of the cell cycle. We have shown that topoisomerase II is the target of casein kinase II (CKII) in yeast by comparison of in vivo and in vitro phosphotryptic peptide maps. Limited proteolysis and probing with domain specific antibodies show that with the exception of a weakly modified residue between aa 660 and aa 1250, all residues modified by CKII are in the last 200 amino acids of yeast topoisomerase II. This C-terminal domain is the least conserved region of the enzyme and truncation of the enzyme shows that it is nonessential for activity in vitro. However, the fully dephosphorylated full-size protein is nearly inactive in decatenation assays, and activity can be restored by phosphorylation by CKII. To reconcile these observations, we propose that the C-terminal region is a negative regulatory domain, counteracted by phosphorylation within the domain itself. To test this hypothesis we have mutagenised 12 potential CKII phosphoacceptor sites in the C-terminus of topoisomerase II and introduced the mutant genes into a yeast strain which has a temperature sensitive top2 gene. The growth of the transformed strains is monitored at nonpermissive temperature to determine whether C-terminal phosphorylation is important for mitotic growth. In addition, we have purified the mutant enzymes to homogeneity for in vitro assays.
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PMID:The regulation of DNA topoisomerase II by casein kinase II. 773 31

Topoisomerase II protein is essential for cell proliferation and is known to exist as a phosphoprotein in cells from both lower and higher eukaryotic species. In this paper, we have investigated the phosphorylation of the alpha isozyme of human topoisomerase II. The topoisomerase II alpha protein was phosphorylated predominantly on serine residues in the human tumor cell lines HeLa and NSCLC-3. Two-dimensional tryptic phosphopeptide mapping studies revealed several sites of phosphorylation in vivo, including a major site that was common to topoisomerase II alpha protein from both HeLa and NSCLC-3 cells. To identify sites of phosphorylation, the regulatory C-terminal domain of human topoisomerase II alpha protein was overexpressed in Escherichia coli as a hexahistidine-tagged fusion protein and purified by nickel chelate chromatography. Tryptic phosphopeptide mapping revealed that casein kinase II phosphorylated the C-terminal domain primarily on 2 serine residues in vitro, which were shown to be sites of modification in vivo. Site-directed mutagenesis studies identified these casein kinase II-specific phosphorylation sites as serine 1524 and serine 1376.
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PMID:Serine 1524 is a major site of phosphorylation on human topoisomerase II alpha protein in vivo and is a substrate for casein kinase II in vitro. 796 67

Mitotic division in yeast requires the activity of topoisomerase II, a DNA topology modifying enzyme that is able to disentangle sister chromatids after DNA replication. Previous work has shown that topoisomerase II is a phosphoprotein in intact yeast cells. We show here that when dephosphorylated in vitro, topoisomerase II is unable to cleave or decatenate kinetoplast DNA. An efficient kinase activity that modifies topoisomerase II on seven major sites was found to copurify with the enzyme purified from yeast. Characterization of this kinase, analysis of phosphotryptic peptides, and studies with a yeast mutant deficient in casein kinase II, indicate that the copurifying kinase is casein kinase II (CKII). Topoisomerase II itself has no self-phosphorylating activity. Modification of topoisomerase II by the copurifying kinase is sufficient to restore decatenation activity after dephosphorylation by alkaline phosphatase. The CKII target sites have been mapped to multiple serine and threonine residues on 4 tryptic fragments within the C-terminal 350 amino acids of yeast topoisomerase II. These results are consistent with a model in which the C-terminal domain of topoisomerase II is a negative regulatory domain that is neutralized by phosphorylation.
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PMID:Casein kinase II copurifies with yeast DNA topoisomerase II and re-activates the dephosphorylated enzyme. 838 77