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Query: EC:5.99.1.2 (topoisomerase)
 9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

cDNA clones of the human TOP1 gene encoding DNA topoisomerase I (EC 5.99.1.2) have been obtained by immunochemical screening of phage lambda libraries expressing human cDNA segments, using rabbit antibodies raised against purified HeLa DNA topoisomerase I. Hybridization patterns between the cloned cDNA sequences and human cellular DNA and cytoplasmic mRNAs indicate that human TOP1 is a single-copy gene. The chromosomal location of the gene has been mapped to the long arm of chromosome 20, in the region q12-13.2, by hybridization of a radioactively labeled TOP1 cDNA probe to human metaphase chromosomes and to a panel of rodent-human somatic hybrids retaining overlapping subsets of human chromosomes.
Proc Natl Acad Sci U S A 1988 Dec
PMID:Human DNA topoisomerase I is encoded by a single-copy gene that maps to chromosome region 20q12-13.2. 284 44

The replication of simian virus 40 origin-containing DNA has been reconstituted in vitro with SV40 large T antigen and purified proteins isolated from HeLa cells. Covalently closed circular DNA (RF I') daughter molecules are formed in the presence of T antigen, a single-stranded DNA binding protein and DNA polymerase alpha-primase complex, together with ribonuclease H, DNA ligase, topoisomerase II, and a double-stranded specific exonuclease that has been purified to homogeneity. The 44-kDa exonuclease-digested oligo(rA) annealed to poly(dT) in the 5'----3' direction. DNA ligase and the 5'----3' exonuclease were essential for RF I' formation. Covalently closed circular duplex DNA and full length linear single-stranded DNA were detected by alkaline gel electrophoresis as products of the complete system. DNA replication in the absence of either DNA ligase or the 5'----3' exonuclease yielded DNA products that were half length (approximately 1500 nucleotides) and smaller Okazaki-like fragments (approximately 200 nucleotides). Hybridization experiments showed that the longer chains were synthesized from the leading strand template, while the small products were synthesized from the lagging strand template. These results suggest that the RNA primers attached to 5' ends of replicated DNA are completely removed by the 5'----3' exonuclease, with the assistance of RNase H.
J Biol Chem 1988 Dec 25
PMID:Complete enzymatic synthesis of DNA containing the SV40 origin of replication. 284 39

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.
Cell 1988 Dec 23
PMID:Localization of a type II DNA topoisomerase to two sites at the periphery of the kinetoplast DNA of Crithidia fasciculata. 284 7

We have studied the three-dimensional folding of the scaffolding in histone H1-depleted chromosomes by immunofluorescence with an antibody specific for topoisomerase II. Two different types of decondensed chromosomes are observed. The majority of the chromosomes are expanded, and the central fluorescence signal is surrounded by a large halo of chromatin. A much smaller number of chromosomes are more compact in length; they contain a smaller halo of chromatin and their scaffolds are not extended but folded into a genuine, quite regular helical coil. This conclusion is based on a three-dimensional structural analysis by optical sectioning. The number of helical coils is related to chromosome length. Surprisingly, sister chromatids have predominantly opposite helical handedness; that is, they are related by mirror symmetry.
Cell 1988 Dec 23
PMID:The metaphase scaffold is helically folded: sister chromatids have predominantly opposite helical handedness. 284 11

Undiluted extracts from eggs or oocytes of Xenopus laevis support the assembly of chromatin with physiologically spaced nucleosomes. Micrococcal nuclease and DNase I digestion experiments show that nucleosome formation as well as supercoiling of circular DNA concomitant to assembly do not require ATP or Mg2+. However these factors are essential for the stability and the physiological spacing of the assembled chromatin. gamma-S-ATP can substitute for ATP in this process. With topoisomers of defined linking number topological interconversions proceed by steps of unity, both in vitro as well as in vivo, indicating that topoisomerase I is dominantly acting in this process. Novobiocin sensitivity occurred only with diluted extracts and was unrelated to an inhibition of topoisomerase II. Finally, nucleosome assembly occurs efficiently on linear DNA although the assembled DNA is less stable than with circular DNA. From these results we propose that mature chromatin is formed in a two-step reaction. In the first step, nucleosome deposition occurs independently of ATP and Mg2+. Thus, nucleosome formation can be uncoupled from their spacing. In this step, topoisomerase activity is involved in the relaxation of the topological constraints generated by chromatin assembly rather than in the process of assembly itself. The second step, requiring ATP and Mg2+, generates properly spaced chromatin.
EMBO J 1988 Dec 20
PMID:Assembly of spaced chromatin involvement of ATP and DNA topoisomerase activity. 285 62

To investigate the role of topoisomerases in the production of sister chromatid exchanges, the effects of inhibitors of type I and II topoisomerases on baseline and mutagen-induced sister chromatid exchanges were compared. V79 cells were treated with VM-26 and m-AMSA, known inhibitors of type II topoisomerase, or with camptothecin, the only known inhibitor of type I topoisomerase. We observed that inhibitors of both type I and II topoisomerases induced high levels of sister chromatid exchanges at 10(-6) M, and that the dose-response curves of these drugs were very similar. A clear heterogeneity in the distribution patterns of exchanges induced by inhibitors of topoisomerases was observed. We believe that this heterogeneity in response to these compounds is due to variation in sensitivity within the cell cycle. We also studied interactions of these agents with mitomycin-C and with PUVA (8-methoxypsoralen + UVA), both cross-linking agents and potent sister chromatid exchange inducers, and with x-rays, an agent that induces high levels of DNA strand breaks. No significant change in exchange levels was observed in interactions between topoisomerase inhibition and the levels induced by the agents studied. We conclude that double-strand break prevalence, known to be increased through inhibition of type II topoisomerase, is not the primary mechanism for induction of sister chromatid exchanges. We further conclude that acute inhibition of type I and type II topoisomerases does not influence substantially the induction of exchanges by other agents.
Cell Biol Toxicol 1986 Dec
PMID:Induction of sister chromatid exchanges by inhibitors of topoisomerases. 285 99

The replication of DNA containing either the polyoma or SV40 origin has been done in vitro. Each system requires its cognate large-tumour antigen (T antigen) and extracts from cells that support its replication in vivo. The host-cell source of DNA polymerase alpha - primase complex plays an important role in discriminating between polyoma T antigen and SV40 T antigen-dependent replication of their homologous DNA. The SV40 origin- and T antigen-dependent DNA replication has been reconstituted in vitro with purified protein components isolated from HeLa cells. In addition to SV40 T antigen, HeLa DNA polymerase alpha - primase complex, eukaryotic topoisomerase I and a single-strand DNA binding protein from HeLa cells are required. The latter activity, isolated solely by its ability to support SV40 DNA replication, sediments and copurifies with two major protein species of 72 and 76 kDa. Although crude fractions yielded closed circular monomer products, the purified system does not. However, the addition of crude fractions to the purified system resulted in the formation of replicative form I (RFI) products. We have separated the replication reaction with purified components into multiple steps. In an early step, T antigen in conjunction with a eukaryotic topoisomerase (or DNA gyrase) and a DNA binding protein, catalyses the conversion of a circular duplex DNA molecule containing the SV40 origin to a highly underwound covalently closed circle. This reaction requires the action of a helicase activity and the SV40 T antigen preparation contains such an activity. The T antigen associated ability to unwind DNA copurified with other activities intrinsic to T antigen (ability to support replication of SV40 DNA containing the SV40 origin, poly dT-stimulated ATPase activity and DNA helicase).
Philos Trans R Soc Lond B Biol Sci 1987 Dec 15
PMID:In vitro replication of DNA containing either the SV40 or the polyoma origin. 289 81

We have been able to map specific DNA fragments at the bases of chromatin loops with the help of a novel extraction procedure by using lithium-3',5'-diiodosalicylate. One such scaffold-attached region (SAR) is found in the non-transcribed spacer in each repeat of the histone gene cluster, on a 657 base pair (b.p.) restriction fragment. Exonuclease III digestion has localized two protein-binding domains on the SAR of the histone cluster. Each covers approximately 200 b.p. and they are separated by a nuclease-accessible region of about 100 b.p. These domains are rich in sequences closely related to the topoisomerase II cleavage consensus. We have studied the scaffold association of three developmentally regulated genes of Drosophila melanogaster: alcohol dehydrogenase (Adh), the homoeotic gene fushi tarazu (ftz) and Sgs-4, a gene encoding one of the glue proteins secreted by third-instar larvae. We find regions attached to the nuclear scaffold (SARS) both 5' and 3' of all three genes, defining small domains ranging from 4.5 to 13 kilobases. In the case of Adh, a gene with two promoters, we find two upstream and two downstream SARS. Those 5' of the gene co-map with regulatory regions for the adult and the larval transcripts, respectively. For Sgs-4, the 5' SAR covers 866 b.p. immediately upstream of the transcript, and encompasses the 200 b.p. regulatory region defined by two deletion mutants that produce little or no Sgs-4 protein. In ftz the 5' SAR is found 4.8 kilobases upstream of the start of transcription within a 2.5 kilobase element required for a high level of ftz expression in the early embryo. Sequence analysis of five upstream SARS reveals clusters of sequences closely related to the cleavage consensus of topoisomerase II. In addition, they contain multiple copies of two sequence motifs: a specific 10 b.p. A-rich sequence, and another 10 b.p. T-rich stretch. In conclusion, the intimate association of the SAR with the upstream/enhancer elements, the presence of clustered sequences highly homologous to the topoisomerase II cleavage consensus, and the localization of topoisomerase II in the scaffold, suggest a structure-function relation between chromosome organization and gene expression.
Philos Trans R Soc Lond B Biol Sci 1987 Dec 15
PMID:Relation of chromosome structure and gene expression. 289 89

Highly torsionally stressed replicative intermediate SV40 DNA molecules are produced when ongoing replicative DNA synthesis is inhibited by aphidicolin, a specific inhibitor of DNA polymerase alpha. The high negative superhelical density of these molecules can be partially released by intercalating drugs such as chloroquine or ethidium bromide. The torsionally stressed replicative intermediates bind to monoclonal anti-Z-DNA antibodies. Electron microscopy of anti-Z-DNA cross-linked to torsionally stressed replicative intermediates shows that the antibody specifically binds close to the replication forks. The superhelical structures are not formed when SV40 DNA replication is inhibited by both aphidicolin and novobiocin, suggesting that a topoisomerase type II-like enzyme is somehow involved in the introduction of torsional strain in replicative intermediate DNA. One interpretation of our data is that fork movement continues to some rather limited extent when SV40 DNA synthesis in replicative chromatin is blocked by aphidicolin. After deproteinization, the exposed single-stranded DNA branches reassociate to form paranemic DNA structures with left-handed helical stretches, while the reduced linking number of the parental strands induces a high negative superhelical density.
EMBO J 1985 Dec 01
PMID:Inhibition of DNA synthesis by aphidicolin induces supercoiling in simian virus 40 replicative intermediates. 300 46

Newly replicated duplex DNA minicircles of trypanosomal kinetoplast DNA are nicked in both their monomeric and catenated topological states, whereas mature ones are covalently sealed. The possibility that nicking may play a role during kinetoplast DNA replication by affecting the topological interconversions of monomeric DNA minicircles and catenane networks was studied here in vitro using Crithidia fasciculata DNA topoisomerase. An enzyme that catalyzes the nicking of duplex DNA circles has been purified to apparent homogeneity from C. fasciculata cell extracts. The native enzyme has a sedimentation coefficient of 6.8 S and was found to be a dimer with a protomer Mr = 60,000. Nicking of kinetoplast DNA networks by the purified enzyme inhibits their decatenation by the Crithidia DNA topoisomerase but has no effect on the catenation of monomeric DNA minicircles into networks. This differential effect on decatenation versus catenation is specific to the purified nicking enzyme. Random nicking of interlocked DNA minicircles has no detectable effect on the reversibility of the topological reaction. The potential role of Crithidia nicking enzyme in the replication of kinetoplast DNA networks in trypanosomatids is discussed.
J Biol Chem 1986 Dec 05
PMID:A nicking enzyme from trypanosomatids which specifically affects the topological linking of duplex DNA circles. Purification and characterization. 302 45


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