共查询到20条相似文献,搜索用时 31 毫秒
1.
Evidences for the function of DNA polymerase-beta in unscheduled DNA synthesis. 总被引:3,自引:1,他引:2
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The activities of DNA polymerase-alpha and -beta isolated from pig spleen were determined at different temperatures and in the presence of different concentrations of inhibitors. The results were compared with parallel estimations of replicative DNA synthesis and UV-induced repair synthesis in spleen cells. In respect to pCMB and aCTP, polymerase-alpha is more sensitive than polymerase-beta and similarly is replication more sensitive than repair. Repair synthesis and the activity of polymerase-beta decreases at temperatures higher than 40 degrees C whereas both replication and the activity of polymerase-alpha are greatly stimulated at elevated temperatures with optima of 45 degrees C (polymerase-alpha) and 41 degrees C (replication). The results favour the hypothesis that polymerase-beta is involved in repair synthesis. 相似文献
2.
A single dose of erythropoietin stimulates DNA synthesis in the spleen of the polycythemic mouse with the maximum effect occurring 48 h after the hormone is administered. The increase in DNA synthesis is accompanied by morphologic evidence of increased erythropoiesis and by increases in the activities per cell of both thymidine kinase and cytoplasmic high molecular weight DNA polymerase-alpha. The activity of low molecular weight DNA polymerase-beta does not change significantly. Spleen cells from mice which had received either erythropoietin or saline 48 h previously were separated into 7 density classes on discontinuous bovine serum albumin gradients. Following the administration of erythropoietin, thymidine incorporation and thymidine kinase activity showed the greatest relative increases per nucleated cell in layers 3, 4 and 5 of the gradient. DNA polymerase-alpha showed the greatest increase in cells of the denser layers 5, 6 and 7. Each layer contained normoblasts and lymphocytes. The less well differentiated erythroid elements constituted a larger proportion of cells in layers of lower density. Increases in the rates of thymidine incorporation were better correlated with increases in thymidine kinase activity than with increases in DNA polymerase activities. Measurement of iron incorporation into heme confirm the morphological impression that the cell type responsible for increased thymidine incorporation and increased DNA polymerase-alpha activity is the young normblast. 相似文献
3.
Nuclei were isolated from monolayer cultures of mouse and human cells using a nonaqueous procedure of cell fractionation in which lyophilized cells were homogenized and centrifuged in 100% glycerol. In previous work we have shown that the nuclear pellet and cytoplasmic supernatant fraction contained 10% or less of the nucleic acids characteristic of the other cell fraction. Aqueous extracts made from fresh cultures and from nonaqueous material at each step of the fractionation procedure were assayed fro DNA polymerase activity. Activities were normalized to DNA contents of extracted material. Specific activity was preserved quantitatively through freezing and drying the cells, but was found to be unstable in glycerol suspensions with approximate half-lives and 1 h at 23 degrees and 4 h at 0-4 degrees. Activities were relatively stable at -25 degrees, however, so that by homogenizing only 15 min at 4 degrees and centrifuging at -25 degrees we preserved approximately 85% of the specific activity of fresh cultures in the nonaqueous nuclear fraction. Sedimentation analyses showed that the nuclear fraction contained both DNA polymerase-alpha and-beta in approximately the proportions expected if all polymerase activities were confined to the nucleus in living cells. DNA polymerase-alpha was found to be more unstable in glycerol suspensions than DNA polymerase-beta. Nuclear location of both activities was found in exponential cultures and in 3T3 mouse cultures synchronized in the G1 and S phases of the cell division cycle. We found no evidence for cytoplasmic factors affecting nuclear polymerase activities. We have concluded that the two major DNA polymerases are nuclear although one, DNA polymerase-alpha, frequently is present as a weakly bound nuclear protein. 相似文献
4.
5.
Dissociated expression of c-myc and a fos-related competence gene during cardiac myogenesis. 总被引:3,自引:1,他引:2
下载免费PDF全文
![点击此处可从《Molecular and cellular biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
M D Schneider P A Payne H Ueno M B Perryman R Roberts 《Molecular and cellular biology》1986,6(11):4140-4143
Cardiac myocytes irreversibly lose their proliferative capacity soon after birth, and cardiac DNA synthesis becomes uncoupled from mitotic division. Therefore, we examined cardiac muscle for developmental down regulation of inducible proto-oncogenes associated with cell proliferation. c-myc mRNA decreased continuously from day 13 of embryonic development and was dissociated from expression of the fos-related gene r-fos, which decreased precipitously between days 3 and 7 after birth. 相似文献
6.
Poly(adenosine diphosphate ribose) polymerase activity and nicotinamide adenine dinucleotide in differentiating cardiac muscle.
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
W C Claycomb 《The Biochemical journal》1976,154(2):387-393
Poly(ADP-ribose) polymerase activity in nuclei isolated from differentiating cardiac muscle of the rat has been characterized and its activity measured during development. Optimum enzyme activity is observed at pH 8.5. Poly(ADP-ribose) polymerase is inhibited by ATP, thymidine, nicotinamide, theophylline, 3-isobutyl-1-methylxanthine and caffeine and stimulated by actinomycin D. The activity measured under optimal assay conditions increases during differentiation of cardiac muscle and is inversely related to the rate of DNA synthesis and to the activities of DNA polymerase alpha and thymidine kinase. When DNA synthesis and the activity of DNA polymerase alpha are inhibited in cardiac muscle of the 1-day-old neonatal rat by dibutyryl cyclic AMP or isoproterenol, the specific activity of poly(ADP-ribose) polymerase measured in isolated nuclei is increased. The concentration of NAD+ in cardiac muscle increases during postnatal development. In the adult compared with the 1-day-old neonatal rat the concentration of NAD+ relative to fresh tissue weight, DNA or protein increased 1.7-fold, 5.2-fold or 1.4-fold respectively. The concentration of NAD+ in cardiac muscle of the 1-day-old neonatal rat can be increased by approx. 20% by dibutyryl cyclic AMP. These data suggest that NAD+ and poly(ADP-ribose) polymerase may be involved with the repression of DNA synthesis and cell proliferation in differentiating cardiac muscle. 相似文献
7.
ts A1S9 mutant cells, derived from wild type WT-4 mouse L-cells, are temperature-sensitive (ts) for DNA synthesis and cell division. We try to determine the cause of the arrest of DNA replication in ts A1S9 cells at the nonpermissive temperature by comparing the modifications induced by the shift of temperature on the activity and the synthesis of DNA polymerase-alpha and DNA primase as a function of time. Forty-seven hours after temperature upshift DNA polymerase-alpha activity of ts A1S9 cells was inhibited by 90% while primase activity was barely detectable. By contrast, the activities of both enzymes increased to a plateau level in WT-4 cultured at either temperature and in ts A1S9 cells grown at the low permissive temperature. Study of the synthesis of DNA polymerase-alpha primase and of the structure of the enzyme complex during cell cycle progression was approached by immunoprecipitation of [35S]-labelled cells, with a specific monoclonal antibody directed against DNA polymerase-alpha. We have found that, irrespective of temperature of cultivation of WT-4 or ts A1S9 cells, this antibody precipitated polypeptides of 220, 186, 150, 110, 68-70, 60, and 48 kDa from cell extracts. With ts A1S9 cells cultivated at 38.5 degrees C for 48 hr the polypeptides of 220 and 186 kDa, associated with alpha-polymerase activity, were considerably more abundant than in the control cells, with a concomitant decline in the polypeptides of 60 and 48 kDa, implicated in primase activity. Thus the inhibition of DNA polymerase-alpha cannot be due to a decreased synthesis of the 186 kDa subunit but to its temperature inactivation. Consistent with a recent asymmetric dimeric model where polymerase-alpha complex and polymerase delta complex synthesize co-ordinately at the replication fork lagging and leading DNA strands, the observed alterations of polymerase-alpha and primase content explain the inhibition of DNA synthesis and the cell cycle arrest of the ts A1S9 cells at the nonpermissive temperature. 相似文献
8.
Delayed and reduced cell replication and diminishing levels of DNA polymerase-alpha in regenerating liver of aging mice 总被引:1,自引:0,他引:1
Kinetics of cell replication was compared in regenerating livers of Mus musculus at ages ranging between 6 and 32 months. Incorporation of [3H]-thymidine into DNA and autoradiographic analysis showed that the maximal extent of DNA replication following partial hepatectomy became delayed with age. Furthermore, the total fraction of parenchymal and nonparenchymal cells in S phase at different intervals during regeneration diminished as mice aged. The specific activity of DNA polymerase-alpha, the putative replicative enzyme, declined progressively during aging. The specific activity of DNA polymerase-beta, the purported repair enzyme, declined to an appreciably lesser extent during the lifespan of the mouse. No evidence was found for the appearance of a specific inhibitor of polymerase-alpha in senescent mouse liver. Also, the bulk of the activities of both hepatic DNA polymerase-alpha and -beta remained localized in the cell nucleus throughout the lifetime of the animal and were mainly associated with chromatin. 相似文献
9.
Growing CV1 cells were infected with simian virus 40 (SV40), and the levels of DNA polymerases-alpha, -beta, and -gamma were analyzed in the cytoplasm, nuclear Triton wash, and nucleus. In the cytoplasmic fraction, the amount of alpha-, beta-, or gamma-polymerase remained unaltered after SV40 infection. The activity of DNA polymerase-alpha increased five- to sixfold in the nuclear Triton wash and threefold in the nuclei and then remained enhanced only inside the nuclei. That of DNA polymerases-beta and gamma increased mostly in the nuclei after infection. These results suggest that DNA polymerase-alpha could be the major enzyme involved in SV40 DNA replication. 相似文献
10.
Differential inhibition of rat liver DNA polymerases in vitro by direct-acting carcinogens and the protective effect of a thiol reducing agent. 总被引:1,自引:0,他引:1
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The direct-acting carcinogens acetoxyacetylaminofluorene, methylnitrosourea, and N-methyl-N'-nitro-N-nitrosoguanidine were tested for their ability to inhibit rat liver DNA polymerase-alpha, -beta, and -gamma activity in vitro. DNA polymerase-alpha was the most sensitive, polymerase-beta was the most resistant, and polymerase-gamma exhibited an intermediate response. When the reactions were reassayed in the presence and absence of dithiothreitol, a thiol reducing agent, it was shown that the inhibition by carcinogens was generally reversible with increasing dithiothreitol, except that polymerase-beta recovered only 80-90% of control values. These and binding data suggest that DNA polymerase-beta, the putative repair enzyme, is highly resistant to carcinogen damage. This resistance may contribute to the retention of normal function and fidelity of the repair enzyme during carcinogen exposure in vivo and to a normal cellular repair. 相似文献
11.
C Terai D B Wasson C J Carrera D A Carson 《Journal of immunology (Baltimore, Md. : 1950)》1991,147(12):4302-4306
Mononuclear phagocytes play a central role in the pathogenesis of chronic inflammatory diseases. It is therefore important to define chemotherapeutically exploitable metabolic pathways that distinguish monocytes from other cell types. Blood monocytes do not synthesize deoxynucleotides de novo, and their transformation to macrophages occurs without cell division. Whether or not monocytes can repair DNA damage, and whether or not DNA repair is necessary for their survival, is unknown. The present experiments demonstrate that normal human monocytes, unlike neutrophils, rapidly repair DNA strand breaks induced by gamma-irradiation. Monocyte extracts contain functional immunoreactive DNA polymerase-alpha. DNA repair synthesis in normal monocytes is blocked by aphidicolin, an inhibitor of DNA polymerase-alpha with respect to dCTP. Aphidicolin is also directly toxic to normal monocytes, but has no effect on nondividing lymphocytes or fibroblasts. Compared to most other cell types, monocytes and macrophages have very low dCTP pools, but abundant deoxycytidine kinase activity. This suggests that dCTP derived from salvage pathways is important for DNA repair in these cells. Consistent with this notion, exogenous deoxycytidine could partially protect monocytes from aphidicolin killing. The unexpected toxicity of aphidicolin toward normal human monocytes may be attributable to their high rate of spontaneous DNA strand break formation, to the importance of DNA polymerase-alpha for DNA repair in these cells, and to their minute dCTP pools. 相似文献
12.
W C Claycomb 《The Journal of biological chemistry》1975,250(9):3229-3235
DNA synthesis and DNA polymerase activity have been measured in terminally differentiating cardiac muscle of the rat. Incorporation of [3H]thymidine into DNA essentially ceases by the 17th day of postnatal development. Cardiac muscle of neonatal rats contains at least two molecular species of DNA polymerase: a 3.5 S DNA polymerase that can be extracted from nuclei with 0.2 m potassium phosphate and a 6 to 8 S soluble cytoplasmic DNA polymerase. The nuclear DNA polymerase in crude extracts has a pH optimum of 9.0 and is more active with native DNA than with denatured DNA as the primer-template. The cytoplasmic DNA polymerase in crude extracts has a pH optimum of 7.5 and is more active with denatured DNA. The activity of the 6 to 8 S cytoplasmic DNA polymerase decreases 80-fold from day 1 to day 17 after birth, which correlates temporally with the reduced rate of DNA synthesis. The activity of the 3.5 S nuclear DNA polymerase remains relatively constant throughout postnatal development. Mixing experiments (assay of neonatal enzyme extracts with adult enzyme extracts) gave additive results, suggesting that the decline in 6 to 8 S DNA polymerase activity apparently is not due to the presence of absence of soluble activators or inhibitors at different times during development. These studies may provide a system which can be used to investigate the control of DNA synthesis and cellular proliferation during the terminal stages of cardiac muscle differentiation. 相似文献
13.
DNA primase from KB cells. Characterization of a primase activity tightly associated with immunoaffinity-purified DNA polymerase-alpha 总被引:21,自引:0,他引:21
A very highly purified fraction of KB cell DNA polymerase-alpha, prepared with a monoclonal antibody, contains DNA primase activity. The primase synthesizes oligonucleotide chains initiated with ATP in a reaction that is resistant to alpha-amanitin and strictly dependent on added template and ribonucleoside triphosphates (rNTPs). In the presence of added dNTPs and M13 DNA template, the primase produces a uniform population of oligoribonucleotides, predominantly hexamers to decamers, that are extended by polymerase-alpha into DNA chains up to 3000 nucleotides long. There is no evidence for nucleotide preferences at RNA/DNA junctions. In the absence of added dNTPs, the oligomeric products are heterogeneous in size and composition and susceptible to cleavage by pancreatic DNase I due to their content of short oligodeoxynucleotide tracts synthesized by primase from trace contaminant dNTPs in the rNTP substrates. The primase and polymerase-alpha activities are distinguishable by several physical and chemical criteria, and the primase reaction is only partially sensitive to two potent, independent monoclonal antibodies that neutralize polymerase-alpha. Although the presence of both primase and polymerase-alpha activities in a highly purified immune complex prepared with a monoclonal antibody argues for their tight physical association, the chemical, physical, and immunological discriminations indicate the two catalytic entities are functionally and structurally distinct. 相似文献
14.
Mononuclear inflammatory cells (MC) isolated from the livers and spleens of mice with chronic graft-vs-host disease (CGVHD) to minor histocompatibility antigens (B10.D2----BALB/c) show defective proliferation when stimulated with Con A and LPS. In turn, both CGVHD liver and spleen cells suppress the proliferation of mitogen-stimulated normal spleen cells in a genetically unrestricted manner. The suppressor activity of CGVHD spleen cells is mediated by plastic nonadherent null (natural suppressor) cells and involves a soluble suppressor factor(s). In contrast, the suppressor activity of CGVHD liver cells is mediated by macrophages (M phi). In the current studies we show that the suppressor activity of CGVHD liver cells is also mediated by soluble factors and compare the roles of prostaglandins and interferon (IFN)-gamma in mediating defective proliferation and suppressor activities of CGVHD liver and spleen MC. Monoclonal antibody to IFN-gamma partially reversed the defective mitogen-stimulated proliferation of CGVHD spleen MC but had no effect on proliferative response of CGVHD liver MC. Indomethacin did not alter the low proliferative response of either CGVHD liver or spleen MC. Anti-IFN-gamma inhibited the ability of CGVHD spleen cells to suppress proliferation of Con A and LPS-stimulated B10.D2 spleen cells. In contrast, anti-IFN-gamma resulted in a small decrease in the ability of liver MC to suppress Con A (but not LPS)-stimulated cell proliferation. Indomethacin decreased the ability of both CGVHD liver and spleen cells to suppress Con A-stimulated proliferation but had inconsistent effects on LPS-stimulated proliferation. These results show that IFN-gamma and prostaglandins partially mediate the suppressor activity of CGVHD spleen MC. The suppressor activity of CGVHD liver MC also involves prostaglandins but is relatively independent of IFN-gamma. 相似文献
15.
The silk gland of Bombyx mori is a terminally differentiated tissue in which DNA replication continues without cell or nuclear division during larval development. DNA polymerase-delta activity increases in the posterior and middle silk glands during the development period, reaching maximal levels in the middle of the fifth instar larvae. The enzyme has been purified to homogeneity by a series of column chromatographic and affinity purification steps. It is a multimer comprising of three heterogeneous subunits, M(r) 170,000, 70,000, and 42,000. An auxiliary protein from B. mori silk glands, analogous to the proliferating cell nuclear antigen, enhances the processivity of the enzyme and stimulates catalytic activity by 3-fold. This auxiliary protein has also been purified to homogeneity. It is a dimer comprised of a single type M(r) 40,000 subunit. Polymerase-delta possesses an intrinsic 3'----5' exonuclease activity which participates in proofreading by mismatch repair during DNA synthesis and is devoid of any primase activity. DNA polymerase-delta activity could be further distinguished from polymerase-alpha from the same tissue based on its sensitivity to various inhibitors and polyclonal antibodies to the individual enzymes. Like DNA polymerase-alpha, polymerase-delta is also tightly associated with the nuclear matrix. The polymerase alpha-primase complex could be readily separated from polymerase-delta (exonuclease) in the purification protocol adopted. DNA polymerase-delta from B. mori silk glands resembles the mammalian delta-polymerases. Considering that both DNA polymerase-delta and -alpha are present in nearly equal amounts in this highly replicative tissue and their close association with the nuclear matrix, the involvement of both the enzymes in the chromosomal endoreplication process in B. mori is strongly implicated. 相似文献
16.
17.
Differentiation of cardiac myocytes after mitogen withdrawal exhibits three sequential states of the ventricular growth response 总被引:9,自引:0,他引:9
下载免费PDF全文
![点击此处可从《The Journal of cell biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
During cardiac myogenesis, ventricular muscle cells lose the capacity to proliferate soon after birth. It is unknown whether this developmental block to mitotic division and DNA replication might involve irreversible repression of the cellular oncogene c-myc. Ventricular myocytes from 2 d-old rats continued to differentiate in vitro during 15 d of mitogen withdrawal, as shown by the formation of cross-striations, increased proportion of the muscle isoenzyme of creatine kinase, stable expression of alpha-cardiac actin and myosin heavy chain mRNAs, and appropriate down-regulation of alpha-skeletal actin mRNA. After mitogen withdrawal for 2 d, serum evoked both DNA synthesis and mitotic division; after 7 d, DNA replication was uncoupled from cell division; after 15 d, DNA synthesis itself was markedly attentuated. These three distinct phenotypic states resemble the sequential properties of growth found in the neonatal rat heart in vivo. Despite failure to induce DNA replication or division after 15 d of mitogen withdrawal, serum elicited both c-myc and alpha-skeletal actin as found during hypertrophy of the intact heart. The results agree with previous evidence that one or more functional pathways that transduce the effects of serum factors may persist in older cardiac muscle cells, and indicate that irreversible down-regulation of c-myc cannot be the basis for the loss of growth responses. 相似文献
18.
Selective stimulation by tri-iodothyronine of myocardial deoxyribonucleic acid polymerase-α in neonatal rats
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Constantinos J. Limas 《The Biochemical journal》1979,180(1):59-67
Three forms of DNA polymerase (alpha, beta and gamma) were separated from isolated rat myocardial cells on the basis of template, pH and ionic requirements, sensitivity to N-ethylmaleimide and position on sucrose gradients. Tri-iodothyronine administration (20mug/100g intraperitoneally) to 3-week-old rats resulted in selective stimulation of DNA polymerase-alpha (198+/-7.1 versus 102+/-5.8pmol of [(3)H]dTMP/30min per mg of protein in untreated controls, P<0.01), with no change in polymerases-beta and -gamma. [(3)H]Thymidine incorporation into myocardial DNA was also enhanced in tri-iodothyronine-treated neonatal rats (132+/-11.2 versus 53+/-4.1c.p.m./mug of DNA in controls, P<0.001). Increased incorporation was associated with an expansion of deoxyribonucleoside 5'-triphosphate pools, especially that of dTTP (24+/-1.6 versus 10+/-1.1pmol/mg of DNA, P<0.01). Neither DNA polymerase activities nor [(3)H]thymidine incorporation were changed in 6-month-old rats in response to tri-iodothyronine. Unstimulated adult myocardial cells had DNA polymerase activities comparable with those in 3-week-old animals, but significantly lower [(3)H]-thymidine incorporation and deoxyribonucleoside triphosphate concentrations. Enhancement of both DNA polymerase-alpha activity and [(3)H]thymidine incorporation in tri-iodothyronine-treated young rats was prevented by concomitant administration of either vinblastine (1mug/g) or daunomycin (2mug/g); actinomycin D (0.1mug/g) or cycloheximide (8mug/g), on the other hand, prevented the increase in [(3)H]thymidine incorporation, but not DNA polymerase-alpha activation. These results demonstrate an age-dependent stimulation of myocardial DNA replication by tri-iodothyronine and suggest an inter-relationship between DNA synthesis and subsequent entry into mitosis. 相似文献
19.
Studies on the purification and properties of a 6.8-S DNA polymerase activity found in calf-thymus DNA polymerase-alpha fraction. 总被引:2,自引:0,他引:2
I P Hesslewood A M Holmes W F Wakeling I R Johnston 《European journal of biochemistry》1978,84(1):123-131
The heterogeneity of calf thymus DNA polymerase-alpha has been further investigated. In particular, an enzyme (enzyme D) which exhibits higher activity on poly(dA) . (dT)10 (A:T = 20:1) compared with that on activated DNA, has been further purified and its properties compared with two other activities of the DNA polymerase-alpha fraction (enzymes A1 and C) which do not show a preference for poly(dA) . (dT)10 over activated DNA. As with A1 and C, enzyme D was shown to have many of the characteristic properties of DNA polymerase-alpha in that it is an acidic protein as judged by its binding to DEAE-cellulose, has a molecular weight of about 140000, does not use a poly (A) . (dT)10 template-initiator complex and is inhibited by N-ethylmaleimide. It exhibits anomalous gel filtration behaviour on Sepharose 6B and it binds relatively weakly to DNA-cellulose compared with DNA polymerase-beta. The extreme sensitivity of enzyme D to inhibtion by N-ethylmaleimide distinguishes it from A1 and C, as does its elution position from a DEAE-cellulose column. On the other hand enzymes C and D are readily inactivated by heating at 45 degrees C unlike enzyme A1. The possible interrelationships of the multiple activities of calf thymus DNA polymerase-alpha are discussed. 相似文献
20.
DNA polymerase activity in a repair-deficient human cell line 总被引:1,自引:0,他引:1
C O Joe J O Norman T R Irvin D L Busbee 《Biochemical and biophysical research communications》1985,128(2):754-759
A human low-density-lipoprotein (LDL) receptor-deficient diploid fibroblast cell line (GM1915) was determined to be short patch competent (DNA polymerase-beta) and long patch deficient (DNA polymerase-alpha) for DNA excision repair. Analysis of DNA from GM1915 cells or from WI38 control cells, following treatment with a mutagen known to initiate long patch excision repair, showed that GM1915 cells exhibited decreased resynthesis of oligonucleotide segments excised during repair. When cells deficient in DNA polymerase-alpha activity were permeabilized to permit LDL entry, repair synthesis immediately increased. These data suggest that DNA polymerase-alpha is not activated by mutagen treatment in GM1915 cells and that introduction of LDL into the cells results in activation of the enzyme. 相似文献