Regulation of Xenopus laevis DNA topoisomerase I activity by phosphorylation in vitro

Kaiserman, H. B.; Ingebritsen, Thomas S.; Benbow, Robert M.

doi:10.1021/bi00409a014

Cited by 64 publications

(49 citation statements)

References 30 publications

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“…Its activity [24-261, but not the amount or stability of the protein [39], varies during the cell cycle. Topoisomerase I is isolated from Novikoff hepatoma cells and Xenopus laevis ovaries as a phosphoprotein and treatment of it with alkaline phosphatase decreases or abolishes its DNA relaxation activity [38,40]. In addition, it is phosphorylated by casein kinase II in vitro resulting in the stimulation of its activity approximately three-fold [38,40,41].…”

Section: Discussionmentioning

confidence: 99%

Protein kinase C phosphorylates DNA topoisomerase I

1989

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The induction of mammalian cell proliferation requires the expression of a specific set of genes. Tumor promoters stimulate cell growth by activating the Ca*+ and phospholipid-dependent protein kinase, protein kinase C (PKC). DNA topoisomerase I, a nuclear enzyme involved in transcription, was phosphorylated by activated PKC in vitro. Phosphorylation by PKC stimulated the DNA relaxation activity of topoisomerase I two-to threefold. Therefore, DNA topoisomerase I is a substrate for PKC-mediated activation by phosphorylation and may serve as a nuclear target of mitogenic signals generated by tumor promoters in vivo.

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Section: Discussionmentioning

confidence: 99%

Protein kinase C phosphorylates DNA topoisomerase I

1989

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“…lOC). A patient serum known to recognize topoisomerase I, a nuclear protein of 110 kDa in Xenopus [41], reacted with the 110-kDa protein in the nuclear but not the cytoplasmic fraction (Fig. 10A).…”

Section: Subcellular Localizationmentioning

confidence: 99%

Purification, primary structure, bacterial expression and subcellular distribution of an oocyte‐specific protein in Xenopus

Rother

Frank

Thomas

1992

European Journal of Biochemistry

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This study defines a novel Xenopus luevis protein (PIOO) that has recently been shown to be recognized by scleroderma patient sera. Using a combination of differential solubility in detergents, hydroxyapatite chromatography and one-dimensional PAGE, PlOO was purified to apparent homogeneity and the amino acid sequence was obtained. An oligonucleotide derived from this sequence was used to clone PlOO cDNA through a polymerase-chain-reaction cloning strategy. The entire PlOO cDNA sequence was determined, identifying a novel 83000-Da protein. Two alleles for PlOO were transcribed in the oocyte, with only one predicted amino acid change between them. Bacterial expression of a clone containing the entire PlOO coding region produced a protein that migrated at a mass 15% greater than that predicted from the amino acid sequence, indicating an aberrant electrophoretic mobility. The mRNA transcript for PlOO was only expressed during the previtellogenic stages of oogenesis (stages I and 11) and was absent from other Xenopus tissues. Similarly, the PlOO protein was found only in Xenopus oocytes and was localized to the cytoplasm of these cells. PlOO irreversibly bound single-stranded-DNA -cellulose but not double-stranded-DNA -cellulose. These data demonstrate the presence of a novel oocyte-specific protein in Xenopus.Autoantibodies in rheumatic diseases target a variety of highly conserved intracellular proteins, including those involved in RNA polymerase I and 111 transcription, heteronuclear RNA splicing, aminoacylation of certain tRNA species, DNA replication, DNA supercoiling and mitosis (review in [l]). The Xenopus oocyte and embryo have provided valuable systems for the characterization of many of these proteins. One example is the ribonucleoproteins targeted by systemic lupus erythematosus patient sera [2]. Antibodies against Sm and RNP were shown to inhibit intron excision from ribosomal genes when injected into germinal vesicles of oocytes, confirming that the RNA-splicing function of these particles demonstrated in vitro also occurred in vivo [3-51. Furthermore, Xenopus Sm and RNP were able to package exogenous small-nuclear RNA from mouse, Drosophila and yeast [6, 71. The Xenopus egg and early embryo were also shown to contain a large store of the U1 small nuclear RNA [6]. Fibrillarin, a ribonucleoprotein associated with U3 small nuclear RNA and recognized by scleroderma patient antibodies, was first cloned and sequenced from a Xenopus cDNA library [8, 91. Other autoantigenic proteins characterized in the Xenopus system include the proliferating cell nuclear antigen and the histones 130-131.Recent experiments in our laboratory have shown that scleroderma sera recognize a 100-kDa protein (P100) which is expressed in Xenopus luevis ovary [14]. Preliminary work also suggests a low level of expression in HeLa cells; however, attempts to clone a 100-kDa protein from a HeLa cell cDNA expression library with such sera have proven to be unsuccessful. In light of the abundance of a 100-kDa protein in X . luevis oocytes...

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“…Similar results were found with purified Top1 from Xenopus laevis (144), Chinese hamster (DC3F/9-OHE) and mouse leukemia cells (L1210) (145), where alkaline phosphatase completely abolished activity, but activity was regained after phosphorylation with Casein kinase II.…”

Section: Post-translational Modifications Of Top1supporting

confidence: 80%

Enzyme Architecture and Flexibility Affect DNA Topoisomerase I Function

Merwe¹

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ii DEDICATION I would like to dedicate this dissertation to my parents, Andries van der Merwe and Marita Esterhuyse for giving me roots so that I can stand strong and to my husband and best friend, Jan Kirsten, for giving me wings so that I can follow my dreams. demonstrate that the conserved core and C-terminal domains dictate the intrinsic enzyme sensitivity to CPT, while it is the functional interactions of the N-terminal and linker domains that regulate enzyme activity in vivo.vi "Since the two chains in our model are intertwined, it is essential for them to untwist if they are to separate. Although it is difficult at the moment to see how these processes occur without everything getting tangled, we do not feel that this objection would be insuperable" (1).In 1953 the scientific duo, Watson and Crick solved the structure of duplex DNA.They found that DNA exists as a double helix and that the bases on each strand were complementary to each other. This helical and complementary character of the DNA double helix represented an elegant solution to the complicated task of housing genetic information. It was further envisioned that to gain access to the information in this molecule, the two strands of the double helix must separate from each other and that the separation will have topological consequences (1). This problem leads to the fundamental need in all cells for a class of enzymes that is able to alter DNA topology, hence DNA topoisomerases. These enzymes are ubiquitous and found in all organisms, including viruses, bacteria, archaebacteria and eukaryotes (2).All DNA topoisomerases alter DNA topology through a cleavage/religation mechanism that employs the chemistry of transesterification. Cleavage is initiated by the Type IA enzymes catalyze DNA strand passage by an "enzyme bridging" mechanism, whereby the enzyme cleaves a single DNA strand and holding onto both DNA ends at the break, bridges the opening through which an intact strand is passed. It does not require an external energy source such as ATP, but Mg(II) is necessary for its relaxation activity.Type IA topoisomerases first bind to a short stretch of single stranded DNA and therefore preferentially relaxes negatively supercoiled or underwound DNA. After binding to the single stranded DNA, the nucleophilic O-4 oxygen of the catalytic Tyr Single molecule experiments have confirmed this model of relaxation by type IA enzymes and further demonstrated that this enzyme is able to increase the linking number of positively supercoiled DNA providing that there is a short stretch of unpaired DNA (11). This demonstrates that the enzyme dictates directionality to the relaxation process.Type IB topoisomerase is distinct from type IA in its mechanism of action as it relaxes DNA by strand rotation. This enzyme binds to duplex DNA and can relax positive or negative supercoils in the absence of energy cofactors or divalent cations. As it relaxes both positive and negative supercoils, the directionality of relaxation is determined by the torsional strain in th...

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Regulation of Xenopus laevis DNA topoisomerase I activity by phosphorylation in vitro

Cited by 64 publications

References 30 publications

Protein kinase C phosphorylates DNA topoisomerase I

Protein kinase C phosphorylates DNA topoisomerase I

Purification, primary structure, bacterial expression and subcellular distribution of an oocyte‐specific protein in Xenopus

Enzyme Architecture and Flexibility Affect DNA Topoisomerase I Function

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