In situ localization of DNA topoisomerase II, a major polypeptide component of the Drosophila nuclear matrix fraction.

Abstract

DNA topoisomerase II has been immunochemically identified on protein blots as a major polypeptide component of the Drosophila nuclear matrix-pore complexlamina fraction. Indirect immunofluorescence analyses of larval cryosections have confirmed the nuclear localization of topoisomerase II in situ. Although apparently excluded from the nucleolus, the topoisomerase protein is otherwise distributed throughout the interior of interphase nuclei. Similar immunocytochemical studies performed with permeabilized whole giant cells from third-instar larval salivary glands have shown topoisomerase II to be largely restricted to the polytene chromosomes. Upon nuclear disassembly during mitosis, the topoisomerase polypeptide appears to redistribute diffusely throughout the cell. Faint immunofluorescent staining of mitotic chromosomes is also observed.The Drosophila melanogaster embryo nuclear matrix-pore complex-lamina (NMPCL) fraction is operationally defined as that material which remains insoluble (pellets at 10,000 x g) during sequential treatments with RNase, DNase, 2% Triton X-100, and 1 M NaCl (1). By phase-contrast microscopy, NMPCL components resemble unfractionated nuclei. However, they are almost entirely devoid of DNA, RNA, phospholipid, and histones. By transmission electron microscopy, these nuclear remnants appear as ovoid or spherical structures surrounded by a limiting layer of fibrous material presumably derived from the nuclear lamina. They also contain an internal meshwork termed the nuclear matrix. Nucleolar remnants can be readily identified within the matrix, while higher-magnification examination of the NMPCL periphery reveals the presence of nuclear pore complexes apparently attached to the lamina. The Drosophila NMPCL is morphologically indistinguishable from fractions similarly derived from a variety of vertebrate nuclei (2-5). It is also similar to the rat liver nuclear pore complex-lamina fraction with regard to elements of the nuclear periphery (6-8).When analyzed by one-dimensional NaDodSO4/PAGE, the Drosophila NMPCL is heterogeneous (1). Two major polypeptides, migrating as a closely spaced doublet in the 74-to 76-kDa region of the gel, have been shown to be antigenically related and to be specifically localized to the nuclear envelope in situ (9). These polypeptides are apparently homologous to the three vertebrate lamins, A, B, and C (10, 11). A third major NMPCL band, migrating in the 170-to 190-kDa region of the NaDodSO4/polyacrylamide gel, has been shown to be composed of two immunochemically distinct polypeptides of nearly identical molecular weight (12 (14) and appears to be homologous to the 190-kDa rat liver nuclear pore complex glycoprotein (15). To date, no quantitatively major NMPCL polypeptide has been definitively localized to the nuclear interior in situ.Recent work in our laboratory on NMPCL ATPases (12, 13) has led to the consideration of DNA topoisomerase II.Type II DNA topoisomerases catalyze the relaxation of superhelical turns in topologically constrained DNA by a d...

Human telomeres are attached to the nuclear matrix.

“…Previously described matrix attachment regions are generally A/T-rich and contain sequences identical or similar to the cleavage site of topoisomerase II, a major component of the nuclear matrix and chromosomal scaffold (Cockerill and Garrard, 1986;Berrios et al, 1985;Earnshaw et al, 1985;. By contrast, human telomeric repeats are G-rich and although topoisomerase II cleavage of telomeric repeats has not been tested, the repeat sequences do not conform the topoisomerase II consensus site (GTNA/TAC/TATTNATNNA/G).…”

DNA Topoisomerase II Is Not Detectable on Lampbrush Chromosomes but Enriched in the Amplified Nucleoli of Xenopus Oocytes

“…One of the best characterized components of the chromosomal scaffold is DNA topoisomerase 11 (topo 11) [4][5][6][7]. Besides its enzymatic activity that creates transient double-strand breaks in duplex DNA and passes DNA strands through one another, the protein has been shown to interact with the MAR and SAR elements [8].…”

“…255 meres containing highly condensed chromatin, while the lateral loops consist of decondensed, transcriptionally active chromatin covered with numerous nascent transcripts. Because lampbrush chromosome architecture resembles so closely the proposed organization of somatic interphase chromatin and mitotic chromosomes, it is tempting to speculate that the same molecular components might be involved in the maintenance of chromatin loops.One of the best characterized components of the chromosomal scaffold is DNA topoisomerase 11 (topo 11) [4][5][6][7]. Besides its enzymatic activity that creates transient double-strand breaks in duplex DNA and passes DNA strands through one another, the protein has been shown to interact with the MAR and SAR elements [8].…”

Localization of specific topoisomerase I interactions within the transcribed region of active heat shock genes by using the inhibitor camptothecin.

“…By using the drug VM26, Rowe et al (38) have shown that topoisomerase II can interact in vivo near the 5' and 3' ends of the hsp70 heat shock gene. Immunofluorescence studies of interphase nuclei and polytene chromosomes of D. melanogaster show a broad distribution of topoisomerase II within the nuclei and chromosomes, respectively (3,25). In contrast, immunofluorescense studies of Drosophila polytene chromosomes have shown that topoisomerase I is restricted to a subset of loci, apparently being recruited to those that are transcriptionally active (15,43).…”