Microinjection of anti-topoisomerase I immunoglobulin G into nuclei of Chironomus tentans salivary gland cells leads to blockage of transcription elongation.

Egyházi, E.; Durban, Egon

doi:10.1128/mcb.7.12.4308

Cited by 36 publications

(18 citation statements)

References 27 publications

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“…Furthermore, several studies have implicated topo I in the maintenance of both nucleolar structure and function (6,(25)(26)(27)(28). The importance of topo I in the nucleolus is highlighted by the finding that yeast topo I deletion mutants exhibit an increase in rDNA recombination (10).…”

Section: -Dddeeddddddddddkplskssksnrkppktmsitgsgekkwdvlimentioning

confidence: 87%

Identification of a Nucleolin Binding Site in Human Topoisomerase I

Bharti

Olson

Küfe

et al. 1996

Journal of Biological Chemistry

114

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DNA topoisomerase I (topo I) is involved in the regulation of DNA supercoiling, gene transcription, and rDNA recombination. However, little is known about interactions between topo I and other nuclear proteins. We used affinity chromatography with a topo I fusion protein to screen U-937 leukemic cell extracts and have identified nucleolin as a topo I-binding protein. Coimmunoprecipitation and other studies demonstrate that the interaction between topo I and nucleolin is direct. Furthermore, deletion analyses have identified the 166 -210-amino acid region of topo I as sufficient for the interaction with nucleolin. Since nucleolin has been implicated in nuclear transport and in a variety of transcriptional processes, the interaction with topo I may relate to the cellular localization of topo I or to the known role of this topoisomerase in transcription.Eukaryotic type I topoisomerases are capable of relaxing both negatively and positively supercoiled DNA (1-3). This function is believed to be important in cellular processes requiring access to DNA, such as transcription and replication, and inhibitors of topo I 1 block these events (4 -8). However, yeast mutants lacking the enzyme exhibit nearly normal growth, and it has been proposed that other cellular topoisomerases may compensate for the lack of topo I (9). The finding that yeast topo I mutants exhibit an increase in rDNA recombination has supported an irreplaceable role for topo I in maintaining the integrity of the rDNA locus (10). Furthermore, several studies have indicated that topo I is located predominately in the nucleolus (11)(12)(13)(14), although the mechanisms related to nucleolar localization of topo I and its unique effects on rDNA are unclear.In the present studies, we used affinity chromatography with an immobilized fusion protein to identify proteins interacting with topo I. We report that nucleolin, a major nucleolar protein, exhibits binding to topo I in both affinity chromatography and coimmunoprecipitation experiments. A region in the amino terminus of topo I has been identified that is sufficient for the interaction with nucleolin. MATERIALS AND METHODS Preparation of Immobilized Fusion Proteins and AffinityChromatography-Escherichia coli strain BLR (Novagen) was utilized for production of topo I fusion proteins. Expression and purification of recombinant GST-fusion protein was as described (15). The bifunctional imidoester dimethylpimelimidate dihydrochloride (Pierce) was used to link the purified fusion proteins or GST alone to glutathione-Sepharose 4B beads (Pharmacia Biotech Inc.). Protein-loaded beads were first washed sequentially with 0.1 M (pH 8.0), O.1 M (pH 9.0), and 0.15 M (pH 9.0) borate buffers. The beads were then incubated for 1 h in 0.15 M borate buffer (pH 9.0) containing 6 g/ml dimethylpimelimidate dihydrochloride. After two washes with 0.1 M borate (pH 8.0), the beads were incubated for 45 min in 0.1 M borate containing 40 mM ethanolamine. The cross-linked beads were then washed sequentially with PBS, 0.2 M glycine-HCl, ...

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Section: -Dddeeddddddddddkplskssksnrkppktmsitgsgekkwdvlimentioning

confidence: 87%

Identification of a Nucleolin Binding Site in Human Topoisomerase I

Bharti

Olson

Küfe

et al. 1996

Journal of Biological Chemistry

114

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“…In addition to binding RNA and DNA (36,37), nucleolin was reported to be a helicase (38) and to function in nucleocytoplasmic transport by binding nuclear localization sequences (NLSs) (39,40). Several lines of evidence indicate that both nucleolin and Top1 are involved in rRNA synthesis and processing (41)(42)(43)(44)(45)(46)(47)(48)(49). To gain insight into the cellular role of the nucleolin-Top1 interaction, we studied both a recombinant form of nucleolin and the Saccharomyces cerevisiae nucleolin ortholog NSR1.…”

Section: Human Top1mentioning

confidence: 99%

“…Thus, nucleolin may organize an rRNA synthesis and processing complex, with the RBDs and GAR domain involved in localizing nucleolin to rDNA transcription sites and the N terminus involved in recruitment of proteins such as Top1 (49). Interestingly, like Top1 (45,46,73,74), nucleolin has been implicated in transcriptional elongation (75). Therefore, it is possible that the nucleolin-Top1 interaction is particularly important in the elongation phase of rDNA transcription.…”

Section: Fig 5 Immunoblotting Analysis Of the Localization Of Ytop1mentioning

confidence: 99%

Role for Nucleolin/Nsr1 in the Cellular Localization of Topoisomerase I

Edwards

Saleem

Shaman

et al. 2000

Journal of Biological Chemistry

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Nucleolin functions in ribosome biogenesis and contains an acidic N terminus that binds nuclear localization sequences. In previous work we showed that human nucleolin associates with the N-terminal region of human topoisomerase I (Top1). We have now mapped the topoisomerase I interaction domain of nucleolin to the N-terminal 225 amino acids. We also show that the Saccharomyces cerevisiae nucleolin ortholog, Nsr1p, physically interacts with yeast topoisomerase I, yTop1p. Studies of isogenic NSR1 ؉ and ⌬nsr1 strains indicate that NSR1 is important in determining the cellular localization of yTop1p. Moreover, deletion of NSR1 reduces sensitivity to camptothecin, an antineoplastic topoisomerase I inhibitor. By contrast, ⌬nsr1 cells are hypersensitive to the topoisomerase II-targeting drug amsacrine. These findings indicate that nucleolin/Nsr1 is involved in the cellular localization of Top1 and that this localization may be important in determining sensitivity to drugs that target topoisomerases. Human Top11 is a nuclear protein involved in the regulation of DNA structure and is the target of an important new class of antineoplastic drugs, the camptothecins (1, 2). Studies in yeast provide convincing evidence that Top1 is the sole cellular target for camptothecins, with this knowledge facilitating attempts to understand the mechanisms by which the drug destroys cells (3-5). Top1 is a monomeric protein that relaxes supercoiled DNA by creating a transient single-strand nick, with this nick involving the covalent attachment of Top1 to the DNA phosphate backbone via a phosphotyrosine bond. Recent structural studies of Top1 and the Top1-DNA covalent complex led to molecular models of the mechanism by which camptothecins inhibit the catalytic activity of Top1 (6 -8). Nevertheless, formation of CPT-Top1-DNA ternary complexes is insufficient to explain the cytotoxic effects of CPT (9 -11). Current models invoke interactions between ternary complexes and replication or transcriptional machinery as being important in conversion of ternary complexes to lethal forms of DNA damage (12, 13). However, it is not known whether physical interactions between Top1 and other proteins (including proteins involved in replication or transcription) are important in the cytotoxic effects of CPT. Recently, an interaction between Top1 and the SV40 T antigen helicase was shown to modulate formation of Top1-CPT-DNA ternary complexes, suggesting that similar interactions between Top1 and cellular helicases may mediate the cytotoxicity of CPT (14). In addition, Top1 rapidly redistributes from the nucleolus to the nucleus or cytoplasm and is degraded after cellular exposure to . These alterations may confer transient cellular resistance to CPT and could be mediated by interactions between Top1 and other proteins.Top1 is known to physically interact with the following proteins: HMG17 (20), casein kinase II (21), RNA polymerase I (22), nucleolin (23), SV40 T antigen (24,25), p53 (26,27), the TATA-binding protein (28), the splicing factors SF2/ASF (2...

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“…The ability of topoisomerase I to relax supercoiled DNA through transient single-stranded DNA cleavage, strand passage, and religation is needed during transcription to relieve superhelical stress (2,3). In addition, topoisomerase I serves as a modulator of transcriptional initiation through physical interaction with transcription factors such as the TATA box-binding protein, which is a constituent of the general transcription factor IID (TFIID) complex (4,5).…”

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confidence: 99%

The interaction between p53 and DNA topoisomerase I is regulated differently in cells with wild-type and mutant p53

Gobert

Składanowski

Larsen

1999

Proc. Natl. Acad. Sci. U.S.A.

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DNA topoisomerase I is a nuclear enzyme involved in transcription, recombination, and DNA damage recognition. Previous studies have shown that topoisomerase I interacts directly with the tumor-suppressor protein p53. p53 is a transcription factor that activates certain genes through binding to specific DNA sequences. We now report that topoisomerase I can be stimulated by both latent and activated wild-type p53 as well as by several mutant and truncated p53 proteins in vitro, indicating that sequence-specific DNAbinding and stimulation of topoisomerase I are distinct properties of p53. These assays also suggest that the binding site for topoisomerase I on p53 is between amino acids 302 and 321. In living cells, the interaction between p53 and topoisomerase I is strongly dependent on p53 status. In MCF-7 cells, which have wild-type p53, the association between the two proteins is tightly regulated in a spatial and temporal manner and takes place only during brief periods of genotoxic stress. In marked contrast, the two proteins are constitutively associated in HT-29 cells, which have mutant p53. These findings have important implications for both cellular stress response and genomic stability, given the ability of topoisomerase I to recognize DNA lesions as well as to cause illegitimate recombination.DNA topoisomerase I is a nuclear enzyme essential for most aspects of nucleic acid metabolism (1). The ability of topoisomerase I to relax supercoiled DNA through transient single-stranded DNA cleavage, strand passage, and religation is needed during transcription to relieve superhelical stress (2, 3). In addition, topoisomerase I serves as a modulator of transcriptional initiation through physical interaction with transcription factors such as the TATA box-binding protein, which is a constituent of the general transcription factor IID (TFIID) complex (4, 5). This property does not require DNA cleavage but is based on protein-protein interactions. Recent results show that topoisomerase I also possesses a proteinkinase activity, which is specific for serine residues of splicing factors containing an Arg-Ser motif. Phosphorylation of these splicing factors is believed to influence gene expression by altering the splice pattern (6, 7).Eukaryotic topoisomerase I is structurally, functionally, and evolutionarily related to site-specific recombinases (8). During catalysis, topoisomerase I forms a covalent DNA-protein complex with the 3Ј end of one DNA strand. This reaction intermediate can religate with either the 5Ј hydroxyl end of the cleaved strand or with a 5Ј hydroxyl end of a heterologous DNA molecule, resulting in recombinant DNA (9, 10). In yeast, increased topoisomerase I activity is accompanied by increased levels of illegitimate recombination, whereas treatment of mammalian cells with camptothecin and other topoisomerase I-directed antitumor drugs leads to sister chromatid exchange and chromosomal aberrations (11,12). This is most likely because camptothecin treatment results in accumulation of covalent ...

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Microinjection of anti-topoisomerase I immunoglobulin G into nuclei of Chironomus tentans salivary gland cells leads to blockage of transcription elongation.

Cited by 36 publications

References 27 publications

Identification of a Nucleolin Binding Site in Human Topoisomerase I

Identification of a Nucleolin Binding Site in Human Topoisomerase I

Role for Nucleolin/Nsr1 in the Cellular Localization of Topoisomerase I

The interaction between p53 and DNA topoisomerase I is regulated differently in cells with wild-type and mutant p53

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