Discrete Localization of Different DNA Topoisomerases in HeLa and K562 Cell Nuclei and Subnuclear Fractions

Monoclonal antibodies raised against DNA topoisomerase I and against topoisomerase II α and β isoforms, which have been previously demonstrated to be highly specific and capable of detecting cell cycle-related variations of the topoisomerase II isoforms (Negri et al., 1992, Exp. Cell Res. 200, 452-459), have been utilized for a fine subcellular localization. Immunocytochemistry by confocal and electron microscopy have been used for a topological and quantitative evaluation of the fine distribution of the different topoisomerases in HeLa and K562 cells. Topoisomerase I and topoisomerase II α are present both in the nucleoplasm and in the nucleolus, though at different relative ratios, while topoisomerase II β is exclusively present at the nucleolar level. This is further confirmed by immunoblotting and immunocytochemical quantitative evaluations performed on purified nuclear matrix fractions obtained from K562 cells. In fact, the amount of topoisomerase I and topoisomerase II α present in the whole cell nuclei is partly lost in isolated nuclei but, while topoisomerase I is further significantly reduced in nuclear matrix preparations, the topoisomerase II α content is only slightly decreased. On the other hand, the great majority of topoisomerase II β is retained in the nuclear matrix and can be detected exclusively in association with the nucleolar remnant. These results are consistent with specific functional roles hypothesized for the different topiosomerase types.     

Association of human DNA topoisomerase IIalpha with mitotic chromosomes in mammalian cells is independent of its catalytic activity.

DNA topoisomerase (topo) II is an essential nuclear enzyme that plays an important role in DNA metabolism and chromosome organization. In the present study, we expressed human topo IIalpha in mammalian cells by fusion to an enhanced green fluorescent protein (EGFP). Decatenation assays indicated that the EGFP-topo IIalpha is catalytically active in vitro. Assays for band depletion, growth inhibition, and cytotoxicity by topo II inhibitors suggested that the fusion protein is also functional in vivo. By following its subcellular localization throughout the cell cycle in living cells, we found that the fusion protein is localized to the nucleus and nucleolus at interphase, and it is bound to chromosomal DNA at every stage of mitosis. Of importance, a mutant EGFP-topo IIalpha, in which the active Tyr 805 is replaced by Phe (Y805F) and is catalytically inactive, still binds to chromosomal DNA throughout the cell cycle like the wild-type enzyme. Together, our results suggest that the ability of topo IIalpha to bind to chromosomal DNA in the cell, a presumed requirement for its structural role, can be separated from its catalytic activity.     

Nucleolar delocalization of human topoisomerase I in response to topotecan correlates with sumoylation of the protein.

DNA topoisomerase (topo) I is an essential nuclear protein and a target for anticancer drug camptothecin derivatives. As a nuclear protein, topo I is concentrated in the nucleolus. However, this nucleolar distribution of topo I is dynamic. It has been shown recently that topo I rapidly moves out of the nucleolus (nucleolar delocalization) in response to topo I inhibitors. In the present study, we demonstrated that nucleolar delocalization of topo I is associated with its conjugation by SUMOs (small ubiquitin-like modifiers) in response to the topo I inhibitor topotecan. Time-course experiments revealed that SUMO-topo I conjugation occurred at as early as 5 min after drug treatment, which was earlier than its observed nucleolar delocalization. Furthermore, heat shock blocked sumoylation of topo I; it also blocked the nucleolar delocalization of topo I fusion proteins. UBC9 is an E2 (ubiquitin carrier protein)-conjugating enzyme essential for sumoylation. Although overexpression of wild-type UBC9 enhanced both sumoylation and nuclear delocalization of topo I, overexpression of a UBC9 dominant negative mutant attenuated topo I sumoylation and its nucleolar delocalization. Taken together, our results suggest that sumoylation of topo I might serve as an addressing tag for its nucleolar delocalization in response to topo I inhibitors.     

Telomeric DNA damage by topoisomerase I. A possible mechanism for cell killing by camptothecin

Topoisomerase I adjusts torsional stress in the genome by breaking and resealing one strand of the helix through a transient covalent coupling between enzyme and DNA. Camptothecin, a specific topoisomerase I poison, traps this covalent intermediate, thereby damaging the genome. Here we examined the activity of topoisomerase I at telomeric repeats to determine whether telomere structures are targets for DNA damage. We show that topoisomerase I is catalytically active in cleaving the G-rich telomeric strand in vitro in the presence of camptothecin but not in cleaving the C-rich strand. The topoisomerase I cleavage site is 5'-TT (downward arrow) AGGG-3' (cleavage site marked by the downward arrow). We also show that endogenous topoisomerase I can access telomeric DNA in vivo and form camptothecin-dependent covalent complexes. Therefore, each telomeric repeat represents a potential topoisomerase I cleavage site in vivo. Because telomere structures are comprised of a large number of repeats, telomeres in fact represent a high concentration of nested topoisomerase I sites. Therefore, more telomeric DNA damage by camptothecin could occur in cells with longer telomeres when cells possess equivalent levels of topoisomerase I. The evidence presented here suggests that DNA damage at telomeric repeats by topoisomerase I is a prominent feature of cell killing by camptothecin and triggers camptothecin-induced apoptosis.     

Quantitation of eukaryotic topoisomerase I reactivity with DNA. Preferential cleavage of supercoiled DNA

A method has been used to quantitate the reaction between eukaryotic type I DNA topoisomerase and topological forms of DNA. This procedure (Trask, D.K., DiDonato, J.D. and Muller, M.T. (1984) Eur. Mol. Biol. Organ. J. 3, 671-676) measures the efficiency of DNA cleavage and concurrent formation of a covalent enzyme/DNA complex. Eukaryotic type I topoisomerases react preferentially by 5-10-fold with supercoiled DNA. The effect of supercoiling is clearly evident in that both the initial rate and final extent of the reaction is elevated. Because the dissociation rate is much lower than the association rate, it is possible to isolate native topoisomerase/DNA complexes. These complexes are comprised of enzyme molecules which are catalytically active when challenged with a second supercoiled DNA substrate. Collectively, the data support the conclusion that a functional intermediate in the reaction sequence is being detected and that the avian topoisomerase I preferentially cleaves supercoiled DNA.     

Metal A and Metal B Sites of Nuclear RNA Polymerases Pol IV and Pol V Are Required for siRNA-Dependent DNA Methylation and Gene Silencing

Plants are unique among eukaryotes in having five multi-subunit nuclear RNA polymerases: the ubiquitous RNA polymerases I, II and III plus two plant-specific activities, nuclear RNA polymerases IV and V (previously known as Polymerases IVa and IVb). Pol IV and Pol V are not required for viability but play non-redundant roles in small interfering RNA (siRNA)-mediated pathways, including a pathway that silences retrotransposons and endogenous repeats via siRNA-directed DNA methylation. RNA polymerase activity has not been demonstrated for Polymerases IV or V in vitro, making it unclear whether they are catalytically active enzymes. Their largest and second-largest subunit sequences have diverged considerably from Pol I, II and III in the vicinity of the catalytic center, yet retain the invariant Metal A and Metal B amino acid motifs that bind magnesium ions essential for RNA polymerization. By using site-directed mutagenesis in conjunction with in vivo functional assays, we show that the Metal A and Metal B motifs of Polymerases IV and V are essential for siRNA production, siRNA-directed DNA methylation, retrotransposon silencing, and the punctate nuclear localization patterns typical of both polymerases. Collectively, these data show that the minimal core sequences of polymerase active sites, the Metal A and B sites, are essential for Pol IV and Pol V biological functions, implying that both are catalytically active.     

Structural insights on the small subunit of DNA topoisomerase I from the unicellular parasite Leishmania donovani

Leishmania donovani, the causative organism of visceral leishmaniasis, contains a unique heterodimeric DNA topoisomerase IB (LdTop1). The catalytically active enzyme consists of a large subunit (LdTop1L), which contains the non-conserved N-terminal end and a phylogenetically conserved core domain, and of a small subunit (LdTop1S) which harbours the C-terminal region with a characteristic tyrosine residue in the active site. Heterologous co-expression of LdTop1L and LdTop1S in a topoisomerase I deficient yeast strain, reconstitutes a fully functional enzyme which can be used for structural studies. The role played by the non-conserved N-terminal extension of LdTop1S in both relaxation activity and CPT sensitivity of LdTop1 has been examined co-expressing the full-length LdTop1L with several deletions of LdTop1S lacking growing sequences of the N-terminal end. The sequential deletion study shows that the first 174 amino acids of LdTop1S are dispensable in terms of relaxation activity and DNA cleavage. It is also described that the trapping of the covalent complex between LdTop1 and DNA by CPT requires a pentapeptide between amino acid residues 175 and 179 of LdTop1S. Our results suggest the crucial role played by the N-terminal extension of the small subunit of DNA topoisomerase I.     

Phosphorylation of mammalian DNA topoisomerase I and activation by protein kinase C

The influence of mammalian DNA topoisomerase I phosphorylation on enzyme activity has been investigated. Dephosphorylation by calf intestine alkaline phosphatase abolished the DNA relaxing activity of DNA topoisomerase I and the sensitivity of the enzyme to its specific inhibitor, camptothecin. DNA topoisomerase I could be reactivated by incubation with purified protein kinase C. DNA topoisomerase I was then able to relax supercoiled DNA processively, like the native enzyme, and to cleave 32P-end-labeled SV40 DNA fragments at the same sequences as the native enzyme in the presence of camptothecin. These results show that active DNA topoisomerase I is a phosphoprotein and suggest a possible regulatory role of protein kinase on topoisomerase I activity and on its sensitivity to camptothecin.     

Localization of a type II DNA topoisomerase to two sites at the periphery of the kinetoplast DNA of Crithidia fasciculata

A type II DNA topoisomerase (topollmt), purified to near homogeneity from the trypanosomatid C. fasciculata has been shown to be localized to the single mitochondrion of these kinetoplastid protozoa. Immunoblots show at least a 10-fold higher level of topollmt (per milligram of protein) in preparations of partially purified mitochondria as compared with those from whole cells. Analyses of type I and type II topoisomerase activities in both mitochondrial and whole cell extracts show a 4- to 5-fold higher specific activity of topollmt in mitochondrial extracts while a nuclear type I topoisomerase has a 4- to 5-fold lower specific activity in the same extract. Immunolocalizations using anti-topollmt antibodies show the enzyme to be present in close association with the mitochondrial DNA networks (kinetoplast DNA or kDNA). This association appears at two distinct locations on opposite sides of the kDNA network.     

Monoclonal antibodies to human DNA topoisomerase I and the two isoforms of DNA topoisomerase II: 170- and 180-kDa isozymes

Several monoclonal antibodies of different isotypes specific to human DNA topoisomerase I, to 170- and 180-kDa DNA topoisomerase II isozymes, were produced and characterized. The specificity of monoclonal antibodies was confirmed by comparison with polyclonal antibodies by Western blot, by immunoprecipitation of enzyme activity, and by immunoprecipitation of DNA topoisomerases with characterized polyclonal antisera. Morphological studies performed by immunofluorescence indicate that the three groups of monoclonal antibodies (MoAbs) stain the nucleus with characteristic patterns, which can be compared with those obtained with polyclonal antibodies. In particular the MoAbs to the 100-kDa DNA topoisomerase I stain the nucleolus and the nucleoplasm; the MoAbs to 170- and 180-kDa DNA topoisomerase II give completely distinct intranuclear patterns: those to the 170-kDa protein stain mainly the nucleoplasm, whereas those to the 180-kDa protein stain only the nucleolus. The two DNA topoisomerase II isozymes clearly exhibit fluctuations in their expression during cell growth: the 170-kDa isozyme is more abundant during the logarithmic phase of growth, while the 180-kDa isozyme is mainly present during the plateau phase of growth.     

Essential roles of the RNA polymerase I largest subunit and DNA topoisomerases in the formation of fission yeast nucleolus

A temperature-sensitive lethal mutant nuc1-632 of Schizosaccharomyces pombe shows marked reduction in macromolecular synthesis and a defective nuclear phenotype with an aberrant nucleolus, indicating a structural role of the nuc1+ gene product in nucleolar organization. We cloned the nuc1+ gene by transformation and found that it appears to encode the largest subunit of RNA polymerase I. We raised antisera against nuc1+ fusion polypeptides and detected a polypeptide (approximately 190 kD and 2 x 10(4) copies/cell) in the S. pombe nuclear fraction. By immunofluorescence microscopy, anti-nuc1+ antibody revealed intense staining at a particular nuclear domain previously defined as the nucleolus. The nucleolar immunofluorescence by anti-nuc1+ was faded in nuc1-632 at restrictive temperature and dramatically diminished in the absence of DNA topoisomerases I and II. Thus active RNA polymerase I appears to be required for the formation of the nucleolus as its major component, and DNA topoisomerases appear to be required for the folding of rDNA and RNA polymerase I molecules into the functional organization of nucleolar genes.     

Rapid and prolonged stalling of human DNA topoisomerase I in UVA-irradiated genomic areas

DNA topoisomerase I appears to be involved in DNA damage and repair in a complex manner. The enzyme is required for DNA maintenance and repair, but it may also damage DNA through its covalently DNA-bound, catalytic intermediate. The latter mechanism plays a role in tumor cell killing by camptothecins, but seems also involved in oxidative cell killing and certain stages of apoptosis. Stalling and/or suicidal DNA cleavage of topoisomerase I adjacent to nicks and modified DNA bases has been demonstrated in vitro. Here, we investigate the enzyme's interactions with UVA-induced DNA lesions inside living cells. We irradiated cells expressing GFP-tagged topoisomerase I with an UVA laser focused through a confocal microscope at confined areas of the nuclei. At irradiated sites, topoisomerase I accumulated within seconds, and accumulation lasted for more than 90 min. This effect was apparently due to reduced mobility, although the enzyme was not immobilized at the irradiated nuclear sites. Similar observations were made with mutant versions of topoisomerase I lacking the active site tyrosine or the N-terminal domain, but not with the N-terminal domain alone. Thus, accumulation of topoisomerase I at UVA-modified DNA sites is most likely due to non-covalent binding to damaged DNA, and not suicidal cleavage of such lesions. The rapid onset of accumulation suggests that topoisomerase I functions in this context as a component of DNA damage recognition and/or a cofactor of fast DNA-repair processes. However, the prolonged duration of accumulation suggests that it is also involved in more long-termed processes.     

Analysis of the chicken DNA fragments that contain structural sites of attachment to the nuclear matrix: DNA-matrix interactions and replication

Ten short DNA fragments have been selected from a library of the nuclear matrix-attached DNA (nmDNA) from chicken erythrocytes by their ability to hybridize with the fraction of chicken replication origins isolated by nascent DNA strand extrusion. The primary structure of these fragments has been determined. Five of the sequences contained a topoisomerase II recognition site. Most of the studied DNA fragments also have a common eight-nucleotide motif, GCAGACCG/A. A sequence-specific DNA-binding protein with a MW of 55 kDa that interacted with this motif has been identified. Some of the cloned DNA fragments promoted an increased level of transient plasmid replication in transfected chicken cells. The ability of plasmid bearing nmDNA fragments to replicate correlated directly with their ability to target plasmids to the nuclear matrix compartment.