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1.
Two forms of topoisomerase I can be purified from Xenopus laevis. A protein with a molecular mass of 165 kDa has been identified as topoisomerase I in ovaries (Richard and Bogenhagen, 1989. J. Biol. Chem. 264, 4704-4709). When a similar purification is performed using liver tissue, topoisomerase I is purified as a 110-kDa protein. Separate rabbit antisera were raised against oocyte and liver topoisomerase I polypeptides. Each antiserum reacts in immunoblotting or immunoprecipitation procedures only with the tissue-specific topoisomerase I polypeptide against which it was generated. The failure of the antiserum raised against liver topoisomerase I to cross-react with the oocyte enzyme suggests that the smaller topoisomerase I is not derived from the 165-kDa oocyte enzyme by proteolysis. X. laevis tissue culture cells lysed and processed in the presence of SDS contain the 110-kDa form of topoisomerase I. The 165-kDa form of topoisomerase I disappears during oocyte maturation in vitro. 相似文献
2.
DNase activity in the presence of Ca2+ + Mg2+, Mg2+ alone, Mn2+ alone, or EDTA, and topoisomerase I activity were measured in nuclear extracts of diethylnitrosamine (DEN)-induced hepatomas, regenerating, fetal, and normal rat livers. In hepatoma tissue, the Ca/Mg-dependent DNase activity was lower than in normal tissue and nearly the same as in fetal liver. In the poorly differentiated hepatomas, Mn-dependent DNase activity was higher than in both moderately and well differentiated ones and than in normal liver tissue. The activity of topoisomerase I in hepatomas and in regenerating liver was lower than in normal liver tissue. 相似文献
3.
Miwako Nishizawa Kazushi Tanabe Taijo Takahashi 《Biochemical and biophysical research communications》1984,124(3):917-924
DNA topoisomerase activity together with the activities of DNA polymerase were detected in a form tightly associated with rat liver nuclear matrices. DNA polymerase activities were solubilized from the nuclear matrices of regenerating rat livers by sonic treatment followed by extraction of these activities with detergent and salt. The predominant activity was mainly α-polymerase as judged from the size determined by sucrose density gradient centrifugation. However, only β-polymerase activity was detected in the matrix of normal rat livers. DNA topoisomerase activity, detected in both regenerating and normal liver nuclear matrices, showed a molecular size of about 4 S in sucrose gradient, and was active in the presence of EDTA. These results suggest that this enzyme belongs to type I topoisomerase. 相似文献
4.
Topoisomerase I is the predominant nuclear protein from avian erythrocytes that can be covalently linked to DNA.
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Nuclear extracts of erythrocytes contain proteins which stably or possibly covalently bind to DNA. These proteins can be detected by an assay which was originally developed to quantify stable binding of topoisomerases to DNA [Trask, DiDonato & Muller (1984) EMBO J. 3, 671-676]. In this report, we show that the number of activities detected by this assay in crude extracts of nuclei is limited predominantly to various forms of topoisomerase I. One form, a 50 kDa protein, copurifies with histone H1. Western blotting experiments suggest that the 50 kDa topoisomerase exists in chromatin along with the 105 kDa form. In addition, the ratio between the high and low-Mr forms is relatively constant in erythrocytes and embryonic fibroblasts. These results imply that the multiple forms are not unique to one tissue setting. 相似文献
5.
Cloning and characterization of the gene for the somatic form of DNA topoisomerase I from Xenopus laevis.
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Two distinct tissue-specific forms of DNA topoisomerase I with M(r) of 165 and 110 kDa have been purified from oocytes and somatic cells respectively of the African frog Xenopus laevis. In this paper, cDNAs encoding a Xenopus topoisomerase I were cloned using PCR primers derived from sequences of yeast and human topoisomerase I. A polypeptide expressed from a portion of the coding sequence was recognized by an antiserum directed against the somatic topoisomerase I that had previously been shown to be unable to cross-react with the oocyte enzyme. Thus, the clone encodes the somatic cell topoisomerase I. An antiserum raised against a synthetic peptide containing the sequence surrounding the active site tyrosine of the somatic topoisomerase I reacts with the enzymes purified from both oocytes and somatic cells, indicating that the two enzymes share some limited sequence homology. RNA blot hybridization showed that oocytes contain an abundant store of somatic topoisomerase I mRNA that is not efficiently polyadenylated in oocytes. This stored RNA contains a consensus cytoplasmic polyadenylation element that is found in a variety of mRNAs that are translationally repressed in oocytes. Microinjection into oocytes of in vitro transcribed mRNA prepared from a Myc-tagged construct of the somatic topoisomerase I sequence is translated to yield a 110 kDa product. This suggests that the oocyte-specific 165 kDa topoisomerase I is not produced by tissue-specific post-translational modification of the somatic topoisomerase I. The oocyte enzyme appears to be produced from a minor mRNA species in oocytes that has not yet been identified. 相似文献
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Topoisomerase function during replication-independent chromatin assembly in yeast. 总被引:1,自引:0,他引:1
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DNA topoisomerases I and II are the two major nuclear enzymes capable of relieving torsional strain in DNA. Of these enzymes, topoisomerase I plays the dominant role in relieving torsional strain during chromatin assembly in cell extracts from oocytes, eggs, and early embryos. We tested if the topoisomerases are used differentially during chromatin assembly in Saccharomyces cerevisiae by a combined biochemical and pharmacological approach. As measured by plasmid supercoiling, nucleosome deposition is severely impaired in assembly extracts from a yeast mutant with no topoisomerase I and a temperature-sensitive form of topoisomerase II (strain top1-top2). Expression of wild-type topoisomerase II in strain top1-top2 fully restored assembly-driven supercoiling, and assembly was equally efficient in extracts from strains expressing either topoisomerase I or II alone. Supercoiling in top1-top2 extract was rescued by adding back either purified topoisomerase I or II. Using the topoisomerase II poison VP-16, we show that topoisomerase II activity during chromatin assembly is the same in the presence and absence of topoisomerase I. We conclude that both topoisomerases I and II can provide the DNA relaxation activity required for efficient chromatin assembly in mitotically cycling yeast cells. 相似文献
8.
C B Chae M C Smith H P Morris 《Biochemical and biophysical research communications》1974,60(4):1468-1474
Chromatin isolated from several well-differentiated rat hepatomas and regenerating liver contains more nonhistone proteins than chromatin of normal liver. Also there are several differences in the electrophoretic patterns of chromosomal nonhistone proteins between hepatomas and normal liver, but not between regenerating or fetal liver and normal liver. 相似文献
9.
Immunopurification and characterization of human alpha-L-iduronidase with the use of monoclonal antibodies. 总被引:3,自引:0,他引:3
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alpha-L-Iduronidase from human liver was purified by a three-step five-column procedure and by immunoaffinity chromatography with a monoclonal antibody raised against purified enzyme. Seven bands identified by staining with Coomassie Blue had molecular masses of 74, 65, 60, 49, 44, 18 and 13 kDa and were present in both preparations of the liver enzyme. However, relative to the immunopurification procedure, alpha-L-iduronidase purified by the five-column procedure was considerably enriched in the 65 kDa polypeptide band. The seven bands were identified by Western-blot analysis with two different monoclonal antibodies raised against alpha-L-iduronidase. The chromatographic behaviour of alpha-L-iduronidase on the antibody column was dependent upon the quantity of enzyme loaded. Above a particular load concentration a single peak of enzyme activity was eluted, whereas at load concentrations below the critical value alpha-L-iduronidase was eluted in two peaks of activity, designated form I (eluted first) and form II (eluted second). The following properties of the two forms of alpha-L-iduronidase were determined. (1) The two forms from liver were composed of different proportions of the same seven polypeptides. (2) When individually rechromatographed on the antibody column, each form from liver shifted to a more retarded elution position but essentially retained its chromatographic behaviour relative to the other form. (3) Forms I and II of liver alpha-L-iduronidase showed no difference in their activities towards disaccharide substrates derived from two glycosaminoglycan sources, heparan sulphate and dermatan sulphate. (4) The native molecular size of forms I and II of liver alpha-L-iduronidase was 65 kDa as determined by gel-permeation chromatography. (5) Immunoaffinity chromatography of extracts of human lung and kidney resulted in the separation of alpha-L-iduronidase into two forms, each with different proportions of the seven common polypeptide species. (6) Lung forms I and II were taken up readily into cultured skin fibroblasts taken from a patient with alpha-L-iduronidase deficiency. Liver forms I and II were not taken up to any significant extent. Lung form II gave intracellular contents of alpha-L-iduronidase that were more than double those of normal control fibroblasts, whereas lung form I gave contents approximately equal to normal control values. We propose that all seven polypeptides are derived from a single alpha-L-iduronidase gene product, and that different proportions of these polypeptides can function as a single alpha-L-iduronidase entity.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
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Yp20 is an abundant 20 kDa chromatin associated protein which has been shown to be related antigenically to genuine Hras products. Using Southwestern blots we have demonstrated that Yp20 is a DNA binding protein. It is also shown that protein Yp20 like protein HM (an abundant thermostable 20 kDa DNA binding protein isolated from mitochondria) and like the 21 kDa autonomously replicating sequence binding factor II (ABFII) is able to introduce superhelical turns into circular relaxed DNA in the presence of DNA topoisomerase I activity. We suggest that this protein may be important for chromatin structure and function. 相似文献
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A N Nikolaenko S I Pavlovich A P Ivanov I N Alekseeva D B Shorokh N V Makogon 《Ukrainski? biokhimicheski? zhurnal》1992,64(1):29-35
Antigens of plasma membranes in hepatocytes from regenerating rat liver were studied. Immunochemical investigation with polyvalent rabbits antiserum against plasma membrane proteins in hepatocytes from regenerating and normal rat liver have shown that liver regeneration processes are accompanied by the increase of proteins number with molecular weight of--80 kDa, 62 kDa, 40 kDa and 27 kDa. It is not excluded that protein with molecular weight of 27 kDa is the tissue-specific peripheral protein. The influence of antibodies against proteins of hepatocytes plasmatic membranes on histostructure of pathologically changed liver tissue has been studied. The data obtained testify to a possibility of participation of the above mentioned proteins in the regulation of rat liver regeneration processes. 相似文献
15.
Elongation by RNA polymerase II on chromatin templates requires topoisomerase activity 总被引:3,自引:2,他引:1
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Mondal N Zhang Y Jonsson Z Dhar SK Kannapiran M Parvin JD 《Nucleic acids research》2003,31(17):5016-5024
16.
Topoisomerase I is preferentially associated with normal SV40 replicative intermediates, but is associated with both replicating and nonreplicating SV40 DNAs which are deficient in histones.
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J J Champoux 《Nucleic acids research》1992,20(13):3347-3352
Based on the use of equilibrium centrifugation in CsCl to separate covalent complexes between topoisomerase I and DNA from protein-free DNA, it was concluded previously that the topoisomerase is preferentially associated with replicating SV40 DNA (Champoux, J. J. 1988. J. Virol. 62:3675-3683). One explanation for the failure to find the enzyme associated with nonreplicating viral DNA is that most of the completed DNA is rapidly sequestered for encapsidation and inaccessible to topoisomerase I. This explanation has been ruled out in the present work by the finding that topoisomerase I in COS-1 cells is also preferentially associated with the replicative form of an SV40 origin-containing plasmid that lacks the genes coding for the virion structural proteins and therefore cannot be encapsidated. Thus it appears that some structural feature of the replicating DNA or the replication complex specifically recruits the topoisomerase to the DNA. SV40 DNA which is produced in the presence of the protein synthesis inhibitor, puromycin, is deficient in histones and as a result lacks normal chromatin structure. Topoisomerase I was found to be associated with SV40 DNA under these conditions whether or not it was replicating. This observation is interpreted as an indication that under normal conditions, chromatin structure limits access of topoisomerase I to the nonreplicating viral DNA. 相似文献
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DNA topoisomerase I from calf thymus is inhibited in vitro by poly(ADP-ribosylation) 总被引:6,自引:0,他引:6
J Jongstra-Bilen M E Ittel C Niedergang H P Vosberg P Mandel 《European journal of biochemistry》1983,136(2):391-396
A slight DNA topoisomerase I activity was detected in highly purified poly(ADP-Rib)polymerase prepared from calf thymus. This copurified activity was found to be suppressed under conditions where the poly(ADP-ribosylation) reaction occurs in the presence of NAD. Purified topoisomerase I from calf thymus was shown to be ADP-ribosylated by poly(ADP-Rib) polymerase purified from the same tissue. Poly(ADP-ribosylation) of topoisomerase I produces an inhibition of the enzymatic activity in parallel to the extent of ADP-ribosylation. The fact that a slight poly(ADP-Rib) polymerase activity was also found to copurify with a topoisomerase I preparation and that topoisomerase I activity can be modified by ADP-ribosylation, may suggest a spatial and functional correlation of these two enzymes in chromatin. 相似文献
19.
Okuhara K Ohta K Seo H Shioda M Yamada T Tanaka Y Dohmae N Seyama Y Shibata T Murofushi H 《Current biology : CB》1999,9(7):341-350
BACKGROUND: Alteration of chromatin structure is a key step in various aspects of DNA metabolism. DNA unwinding factors such as the high mobility group (HMG) proteins are thought to play a general role in controlling chromatin structure and a specific role in controlling DNA replication. For instance, in the in vitro simian virus 40 replication system, minichromosomes containing HMG-17 replicate more efficiently than those without it, suggesting that HMG-17 enhances the rate of replication of a chromatin template by unfolding the higher-order chromatin structure. At present, however, only limited data suggest an involvement of DNA unwinding factors in DNA replication. RESULTS: We purified from Xenopus eggs a novel heterodimeric factor, termed DNA unwinding factor (DUF), that consists of 87 kDa and 140 kDa polypeptides. DUF unwinds closed-circular duplex DNA in the presence of topoisomerase I, but it does not possess a DNA gyrase activity: it does not introduce negative supercoils into DNA at the expense of ATP hydrolysis. Cloning and sequencing of the cDNAs encoding the two polypeptides revealed that the 87 kDa polypeptide is homologous to a mammalian HMG protein, T160/structure-specific recognition protein. The 140 kDa polypeptide is homologous to yeast Cdc68, a protein that controls the expression of several genes during the G1 phase of the cell cycle by modulating chromatin structure. Immunodepletion of DUF from Xenopus egg extracts drastically reduced the ability of the extract to replicate exogenously added sperm chromatin or plasmid DNA. CONCLUSIONS: We propose that DUF plays a role in DNA replication in Xenopus egg extracts. 相似文献
20.
Glyoxalase I in regenerating mouse liver exposed to carcinogens 总被引:1,自引:0,他引:1
Glyoxalase I is the first component of glyoxalase system which is involved in detoxification of alpha-ketoaldehydes and converts them to nontoxic substances. This study reports changes in Glyoxalase I activity in relation to DNA synthesis in regenerating liver treated with two polycyclic aromatic hydrocarbons - 7,12-dimethylbenz (a) anthracene and benzo(a)pyrene. Livers after partial hepatectomy show consistent increase in Gly. I which reaches to its peak at 24 hr after surgery. [3H]Thymidine incorporation into DNA also follows the same trend as does Gly. I in regenerating liver. Both the carcinogens have significantly reduced the activity of Gly. I and DNA biosynthesis when compared with untreated partially hepatectomized control livers. The study reveals that though regenerating liver has been considered as an experimental model for neoplasia, unlike tumors (where Gly. I is either absent or in undetectable quantities) it possesses more Gly. I than in normal liver. On the other hand, preneoplastic liver during initiation (in regenerating liver treated with carcinogens, initiation is expected to occur) has very low activity. This suggests that Gly. I is not only involved in controlling growth but possibly is involved in some other phenomenon which is somehow depressed in preneoplastic and cancerous tissues. 相似文献