Identification of a Nucleolin Binding Site in Human Topoisomerase I

Abstract

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, ...

A Novel Nuclear Localization Signal in Human DNA Topoisomerase I

“…Because nucleolin is a multi-function protein, acting as a shuttling protein between cytoplasm and nucleus, it would be reasonable to postulate that such interactions may facilitate its nucleolar localization. Interestingly, aa 166 -210 of topo I represent the minimal sequence for full binding activity to nucleolin (43). Our results indicate that the sequence for nucleolar localization appears to overlap with the nucleolin binding sequence, because aa 157-199 are important for nucleolar accumulation.…”

“…It has been shown that topo I and nucleolin are localized in a similar region of the nucleolus (42). Moreover, topo I interacts physically with nucleolin (43). Because nucleolin is a multi-function protein, acting as a shuttling protein between cytoplasm and nucleus, it would be reasonable to postulate that such interactions may facilitate its nucleolar localization.…”

A conserved motif in transmembrane helix 1 of diphtheria toxin mediates catalytic domain delivery to the cytosol

“…GST and the fusion protein GST-DT140-271 were expressed in E. coli strain BL-21(DE-3) transformed with either pGEX-T4-1 or pGEX-DT-T1 and purified as described in ref. 22. Protein concentrations were determined by the modified Bradford reagent (BioRad).…”

Mechanisms of resistance to topoisomerase I-targeting drugs

“…The Top1-binding protein nucleolin may recruit Top1 to the nucleolus as a result of the high rate of transcription in this region (Bharti et al, 1996). Studies of a nucleolin orthologue in yeast, Nsr1p, indicate that the absence of this protein is associated with relocalization of Top1 from a predominantly nucleolar localization to a diffuse nuclear localization .…”