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1.
H Park  R Davis    T S Wang 《Nucleic acids research》1995,23(21):4337-4344
The status of Schizosaccharomyces pombe (fission yeast) DNA polymerase alpha was investigated at different stages of the cell cycle. S.pombe DNA polymerase alpha is a phosphoprotein, with serine being the exclusive phosphoamino acid. By in vivo pulse labeling experiments DNA polymerase alpha was found to be phosphorylated to a 3-fold higher level in late S phase cells compared with cells in the G2 and M phases, but the steady-state level of phosphorylation did not vary significantly during the cell cycle. Tryptic phosphopeptide mapping demonstrated that the phosphorylation sites of DNA polymerase alpha from late S phase cells were not the same as that from G2/M phase cells. DNA polymerase alpha partially purified from G1/S cells had a different mobility in native gels from that from G2/M phase cells. The partially purified polymerase alpha from G1/S phase cells had a higher affinity for single-stranded DNA than that from G2/M phase cells. Despite the apparent differences in cell cycle-dependent phosphorylation, mobility in native gels and affinity for DNA, the in vitro enzymatic activity of the partially purified DNA polymerase alpha did not appear to vary during the cell cycle. The possible biological significance of these cell cycle-dependent characteristics of DNA polymerase alpha is discussed.  相似文献   

2.
Recent work has shown that macrophage-mediated cytostatic activity inhibits cell cycle traverse in G1 and/or S phase of the cell cycle without affecting late S, G2, or M phases. The present report is directed at distinguishing between such cytostatic effects on G1 phase or S phase using the accumulation of DNA polymerase alpha as a marker of G1 to S phase transition. Quiescent lymphocytes stimulated with concanavalin A undergo a semisynchronous progression from G0 to G1 to S phase with a dramatic increase in DNA polymerase alpha activity between 20 and 30 hr after stimulation. This increase in enzyme activity was inhibited, as was the accumulation of DNA, when such cells were cocultured with activated murine peritoneal macrophages during this time interval. However, if mitogen-stimulated lymphocytes were enriched for S-phase cells by centrifugal elutriation and cocultured with activated macrophages for 4-6 hr, DNA synthesis was inhibited but the already elevated DNA-polymerase activity was unaffected. Similar results were obtained when a virally transformed lymphoma cell line was substituted as the target cell in this assay. These results show that both G1 and S phase of the cycle are inhibited and suggest that inhibition of progression through the different phases may be accomplished by at least two distinct mechanisms.  相似文献   

3.
4.
Cell cycle-dependent phosphorylation of human DNA polymerase alpha   总被引:13,自引:0,他引:13  
The expression of DNA polymerase alpha, a principal chromosome replication enzyme, is constitutive during the cell cycle. We show in this report that DNA polymerase alpha catalytic polypeptide p180 is phosphorylated throughout the cell cycle and is hyperphosphorylated in G2/M phase. The p70 subunit is phosphorylated only in G2/M phase. This cell cycle-dependent phosphorylation is due to cell cycle-dependent kinase(s) and not to phosphatase(s). In vitro evidence indicates the involvement of p34cdc2 kinase in the mitotic phosphorylation of DNA polymerase alpha. Tryptic phosphopeptide maps demonstrate that peptides phosphorylated in vitro are identical to those phosphorylated in vivo. DNA polymerase alpha from mitotic cells is found to have lower affinity for single-stranded DNA than does polymerase alpha from G1/S phase cells. These results imply that the mitotic phosphorylation of polymerase alpha may affect its physical interaction with other replicative proteins and/or with DNA at the replication fork.  相似文献   

5.
Two heat-sensitive (arrested in G1 at 39.5 degrees C) and two cold-sensitive (arrested in G1 at 33 degrees C) clonal cell-cycle mutants of the murine P-815-X2 mastocytoma line were tested for DNA polymerase alpha, beta and gamma activities. After transfer of mutant cells to the respective nonpermissive temperature, DNA polymerase alpha activities decreased more slowly than relative numbers of cells in S phase. Furthermore, numbers of DNA-synthesizing cells decreased to near-zero levels, whereas polymerase alpha activities in arrested cells were as high as 15-40% of control values. After return of arrested cells to the permissive temperature, polymerase alpha activities increased essentially in parallel with relative numbers of cells in S phase. In contrast to the changes in thymidine kinase (Schneider, E., Müller, B. and Schindler, R. (1983) Biochim. Biophys. Acta 741, 77-85), the decrease of polymerase alpha during entry of cells into proliferative quiescence thus appears to be under rather relaxed control, while after return of arrested cells to the permissive temperature the increase in polymerase alpha is tightly coupled with reentry of cells into S phase. For DNA polymerase beta and gamma activities, no obvious correlation with changes in the proliferative state of cells was detected.  相似文献   

6.
To asses the possible roles of the two active forms of mouse DNA polymerase alpha: primase--DNA-polymerase alpha complex (DNA replicase) and DNA polymerase alpha free from primase activity (7.3S polymerase), in nuclear DNA replication the correlation of their activity levels with the rate of nuclear DNA replication was determined and a comparison made of their catalytic properties. The experiments using either C3H2K cells, synchronized by serum starvation, or Ehrlich culture cells, arrested at the S phase by aphidicolin, showed DNA replicase to increase in cells in the S phase to at least six times that of the G0-phase cells but 7.3S polymerase to increase but slightly in this phase. This increase in DNA replicase activity most likely resulted from synthesis of a new enzyme, as shown by experiments using a specific monoclonal antibody, aphidicolin and cycloheximide. Not only with respect to the presence or absence of primase activity, but in other points as well the catalytic properties of these two forms were found to differ; DNA replicase preferred the activated calf thymus DNA with wide gaps of about 100 nucleotides long as a template-primer, while the optimal gap size for 7.3S polymerase was 40-50 nucleotides long. Size analysis of the products synthesized on M13 single-stranded circular DNA with a single 17-nucleotide primer by DNA replicase and 7.3S polymerase suggested the ability of DNA replicase to overcome a secondary structure formed in single-stranded DNA to be greater than that of 7.3S polymerase.  相似文献   

7.
K Kishi 《Mutation research》1987,176(1):105-116
It has been shown that certain types of DNA lesions induced by an S-dependent clastogen are converted to chromosome-type aberrations when their repair is inhibited in the G1 phase of the cell cycle. The purpose of the present study was to investigate which kinds of repair inhibitors have the ability to induce chromosome-type aberrations in cells having DNA lesions and which kinds of DNA lesions will be converted to chromosome-type aberrations when their repair is inhibited. For this purpose, human peripheral blood lymphocytes, which were treated with a clastogen in their G0 phase, were post-treated with one of several kinds of repair inhibitors in the G1 phase, and resulting frequencies of both chromosome-type and chromatid-type aberrations as well as of sister-chromatid exchanges (SCEs) were compared with those of the control cultures: chromatid-type aberrations and SCEs were adopted as cytogenetic indicators of lesions remaining in S and G2 phases. Chemicals used for the induction of DNA lesions were 4-nitroquinoline 1-oxide (4NQO), methyl methanesulfonate (MMS) and mitomycin C (MMC); inhibitors used were excess thymidine (dThd), caffeine, hydroxyurea (HU), 5-fluoro-2'-deoxyuridine (FdUrd), 1-beta-D-arabinofuranosylcytosine (ara C), 9-beta-D-arabinofuranosyladenine (ara A), 1-beta-D-arabinofuranosylthymine (ara T) and aphidicolin (APC). Induction of chromosome-type aberrations was observed in cells pretreated with 4NQO or MMS followed by ara C, ara A, ara T or APC, whereas other combinations of a clastogen and an inhibitor did not induce them. Among the inhibitors, ara C alone induced chromosome-type aberrations in cells without pretreatment. Chromatid-type aberrations were increased only in cells pretreated with MMC and their frequency was enhanced further by post-treatment with ara C. All of the clastogens used in the present experiments induced SCEs. Most inhibitors did not modify the SCE frequencies except for ara C which synergistically increased the frequency in MMC-treated cells. The present study offers further evidence that the lesions responsible for chromosome-type aberrations are those which are repaired quickly, and that they are converted to chromosome-type aberrations when repair by polymerase alpha is inhibited. The effects of ara C on MMC-induced lesions are considered residual effects of ara C treatment in the S or G2 phases rather than repair inhibition in the G1 phase.  相似文献   

8.
A reversible arrest point in the late G1 phase of the mammalian cell cycle   总被引:18,自引:0,他引:18  
The effects of two different cell cycle inhibitors on the proliferation of human lymphoblastoid cells have been analyzed by flow cytometric techniques. Mimosine, a plant amino acid, reversibly blocks the cell cycle at a point which occurs roughly 2 h before the arrest mediated by aphidicolin, an inhibitor of DNA polymerase alpha activity, which defines the G1/S phase boundary. The levels of thymidine kinase mRNA, which increase at the onset of S phase, are higher in cells blocked with aphidicolin than in cells treated with mimosine whereas the opposite results are obtained in the case of p53 mRNA levels, which are known to be maximal in the late G1 phase. These results indicate that mimosine inhibits cell cycle traverse in the late G1 phase prior to the onset of DNA synthesis and identifies a previously undefined reversible cell cycle arrest point.  相似文献   

9.
When Normal Rat Kidney cells are allowed to reenter the cell cycle after quiescence they start to replicate DNA around 12 h, reaching a maximum at 20 h. Activation of DNA polymerase alpha parallels the increase in DNA synthesis. The addition of two different anti-calmodulin drugs, trifluoroperazine (7.5 microM) or W13 (10 micrograms/ml), to the media at 4 h after proliferative activation, inhibits DNA synthesis by 55% and 80%, respectively. The blockade of calmodulin produced by trifluoroperazine allows the cells to progress through G1 phase but stops progression through S phase as determined by 5-Bromo deoxyuridine labeling. Both anti-calmodulin drugs also inhibit by more than 50% the increase in DNA polymerase alpha activity observed at 20 h. These results indicate that a calmodulin-dependent event, essential for the activation of DNA polymerase alpha and subsequently for DNA replication, is produced during G1. Therefore, the control of DNA polymerase alpha activation is one of the ways by which calmodulin is regulating the progression of NRK cells through S phase.  相似文献   

10.
11.
tsFT20 cells, which have temperature-sensitive DNA polymerase alpha-activity, were characterized mainly at the cellular level. The cells lost their ability to synthesize DNA immediately after a shift to non-permissive temperature. The extent of decrease in the activity of DNA polymerase alpha in whole-cell extracts was the same as that of the decrease in the DNA replication ability determined by [3H]thymidine incorporation. At 39 degrees C, tsFT20 cells lost most of their colony-forming ability in one doubling time (16 h). The cells could not grow at higher than 38 degrees C, but could grow at 37 degrees C. When tsFT20 cells were synchronized at the G1/S boundary and incubated at 39 degrees C, they could not complete the S phase, ceasing cell cycle progression in mid-S phase. A temperature shift (33 degrees C----39 degrees C) experiment indicated that the whole S phase was temperature-sensitive, whereas the G2 and M phases were not. These results confirmed that DNA polymerase alpha plays a key role in DNA replication in mammalian cells.  相似文献   

12.
13.
The intracellular events which are involved in controlling the G1 to S phase transition during the eucaryotic cell cycle are important to define in order to understand the mechanisms by which mitogenic and growth arrest-inducing agents control cell growth. Because a change in protein kinase activity is associated with the initial response of cells to mitogenic stimulants and growth factors, we used a kinase renaturation assay to identify specific protein kinases which are modulated as human T cells make the G1 to S phase transition after mitogenic stimulation with lectin. We identified four protein serine/threonine kinases of 180, 97, 85, and 38 kilodaltons which are increased in activity as these cells enter S phase. A-55 kDa serine/threonine kinase (PK55) was shown to have maximal activity during G0 and its activity was reduced by 95% upon movement into S phase. PK55 is inducible in human T cells by removal of interleukin 2 and low serum incubation which arrests cells in G1 phase, indicating that it is closely associated with G1 phase growth arrest. Furthermore, a similar PK55 activity was induced upon growth arrest in HL-60 cells treated with dimethyl sulfoxide and in Daudi cells treated with interferon alpha. Because the cAMP-dependent protein kinase (PK-A) family has been shown to be antiproliferative to lectin stimulated T cells, we were interested in determining whether PK55 was in fact an isozyme of PK-A. Comparative analysis using a specific peptide inhibitor of PK-A activity revealed that PK55 is catalytically distinct from PK-A. This data suggest that increases in PK55 may be associated with the growth-arrested state and further that PK55 is distinct from PK-A.  相似文献   

14.
The premature chromosome condensation (PCC) technique was used to study several factors that determine the yield of chromosome fragments as observed in interphase cells after irradiation. In addition to absorbed dose and the extent of chromosome condensation at the time of irradiation, changes in chromosome conformation as cells progressed through the cell cycle after irradiation affected dramatically the yield of chromosome fragments observed. As a test of the effect of chromosome decondensation, irradiated metaphase Chinese hamster ovary (CHO) cells were allowed to divide, and the prematurely condensed chromosomes in the daughter cells were analyzed in their G1 phase. The yield of chromosome fragments increased as the daughter cells progressed toward S phase and chromosome decondensation occurred. When early G1 CHO cells were irradiated and analyzed at later times in G1 phase, an increase in chromosome fragmentation again followed the gradual increase in chromosome decondensation. As a test of the effect of chromosome condensation, G0 human lymphocytes were irradiated and analyzed at various times after fusion with mitotic CHO cells, i.e., as condensation proceeded. The yield of fragments observed was directly related to the amount of chromosome condensation allowed to take place after irradiation and inversely related to the extent of chromosome condensation at the time of irradiation. It can be concluded that changes in chromosome conformation interfered with rejoining processes. In contrast, resting chromosomes (as in G0 lymphocytes irradiated before fusion) showed efficient rejoining. These results support the hypothesis that cytogenetic lesions become observable chromosome breaks when chromosome condensation or decondensation occurs during the cell cycle.  相似文献   

15.
DNA polymerase III holoenzyme has been purified from Escherichia coli HMS-83, using, as an assay, the conversion of coliphage G4 single-stranded DNA to the duplex replicative form. The holoenzyme consists of at least four different subunits: alpha, beta, gamma, and delta of 140,000, 40,000, 52,000, and 32,000 daltons, respectively. The alpha subunit is DNA polymerase III, the dnaE gene product. The holoenzyme has been resolved by phosphocellulose chromatography into an alpha - gamma - delta complex and a subunit beta (copolymerase III*); neither possesses detectable activity in the G4 system but together reconstitute holoenzyme-like activity. The alpha - gamma - delta complex has been further resolved to yield a gamma - delta complex which reconstitutes alpha - gamma - delta activity when added to DNA polymerase III. The gamma - delta complex contains a product of the dnaZ gene and has been purified from a strain which contains a ColE1-dnaZ hybrid plasmid.  相似文献   

16.
As described previously, a natural product isolated from fungus (Acremonium sp.), dehydroaltenusin, is an inhibitor of mammalian DNA polymerase alpha in vitro [Y. Mizushina, S. Kamisuki, T. Mizuno, M. Takemura, H. Asahara, S. Linn, T. Yamaguchi, A. Matsukage, F. Hanaoka, S. Yoshida, M. Saneyoshi, F. Sugawara, K. Sakaguchi, Dehydroaltenusin, a mammalian DNA polymerase alpha inhibitor, J. Biol. Chem. 275 (2000) 33957_33961]. In this study, we investigated the interaction of dehydroaltenusin with lipid bilayers using an in vitro liposome system, which is a model of the cell membrane, and found that approximately 4% of dehydroaltenusin was incorporated into liposomes. We also investigated the influence of dehydroaltenusin on cultured cancer cells. Dehydroaltenusin inhibited the growth of HeLa cells with an LD50 value of 38 microM, and as expected, S phase accumulation in the cell cycle. The total DNA polymerase activity of the extract of incubated cells with dehydroaltenusin was 23% lower than that of nontreated cells. Dehydroaltenusin increased cyclin E and cyclin A levels. In the analysis of the cell cycle using G1/S synchronized cells by employing hydroxyurea, the compound delayed both entry into the S phase and S phase progression. In a similar analysis using G2/M synchronized cells by employing nocodazole, the compound accumulated the cells at G1/S and inhibited entry into the S phase. Thus, the pharmacological abrogation of cell proliferation by dehydroaltenusin may prove to be an effective chemotherapeutic agent against tumors.  相似文献   

17.
Resting B cells enter and progress through the G1 phase of the cell cycle in response to low concentrations (1 to 5 micrograms/ml) of anti-IgM antibodies. Commitment to enter S phase requires the presence of a fivefold to 50-fold higher concentration of anti-IgM. These and other results strongly suggest that two separately controlled events are involved in B cell activation. The current studies demonstrate that B cells incubated with high concentrations of anti-IgM from the initiation of culture become independent of additional anti-IgM approximately 10 hr before entry into S phase. We have designated this anti-IgM independent portion of the G1 phase of the cell cycle as G1 beta, whereas the earlier phase is referred to as G1 alpha. Furthermore, low concentrations of anti-IgM are sufficient for progress through early portions of G1 alpha, but high concentrations are required for the last 4 to 8 hr (G1 alpha') if the cells are to go through the rest of the cell cycle. Removal of anti-IgM at any time during G1 alpha causes prompt cessation of the size enlargement that accompanies progress through G1. Such cells retain their size and their relative place in G1 for periods of at least 17 hr and recommence movement through G1 alpha phase when anti-IgM is readded. Thus, B cells may exist in states of partial activation and must possess a mechanism to integrate the amount of stimulatory signal they have received; they enter a commitment period for S phase only when that signal passes some threshold value.  相似文献   

18.
We have examined the kinetic relationship between the rate of entry into the S phase in human diploid fibroblast-like (HDFL) monokaryon cells and (1) the concentration of DNA polymerase alpha activity and (2) the cell volume. In the former studies, a first-order dependence between the rate of entry into the S phase and the concentration of DNA polymerase alpha activity was observed, consistent with the enzyme, or a coregulated factor, being rate limiting for this metabolic process. Examination of the nature of the dependence of the rate of entry into the S phase upon cell volume revealed a more complex relationship. The results obtained in studies with synchronized cultures are consistent with the presence of two to three rate-limiting reactants when cell volume is the independent variable. Studies with asynchronous HDFL cell cultures revealed that the smallest cells in the G1 population, presumably the early G1 cells, enter the S phase at an increasing rate as a function of cell volume up to a certain size, beyond which the cells enter at a decreasing rate similar to that observed in the studies with the synchronized cultures. Similar studies examining the relationship between cell volume and the rate of entry into S phase in three established immortal cell lines revealed positive correlation between the rate of entry into S phase and cell volume throughout the size range of the G1 population. This latter observation suggests that the factors involved in the initiation of the S phase may be present in concentrations that are not rate limiting in immortal cell lines.  相似文献   

19.
Immunocytochemical localization of chick DNA polymerases alpha and beta +   总被引:4,自引:0,他引:4  
An immunofluorescent method using specific antibodies was employed to detect DNA polymerases alpha and beta in chick cells. With monoclonal antibodies produced by four independent hybridoma clones, most of the DNA polymerase alpha was shown to be present in nuclei of cultured chick embryonic cells. With a polyclonal, but highly specific, antibody against DNA polymerase beta, this enzyme was also shown to be present in nuclei. DNA polymerase alpha was detected in proliferating cells before cell contact and in lesser amount in resting cells after cell contact, indicating that its content is closely correlated with cell proliferation. On the other hand, similar amounts of DNA polymerase beta were detected in proliferating and resting cells. Furthermore, DNA polymerase beta was detected in nuclei of most cells, while DNA polymerase alpha was detected only in large round nuclei in seminiferous tubules of chick testis. DNA polymerase alpha is presumably present in cells that are capable of DNA replication, and during the cell cycle it seems to remain in the nuclei during the G1, S, and G2 phases, but to leave from condensed chromatin for the cytoplasm during the mitotic phase.  相似文献   

20.
Recently we reported there were at least four types of G0 or G0-like proteins in bovine brain membranes based on their elution profiles from Mono Q columns and their immunological reactivities; one of the proteins was purified as an alpha-monomeric form, and the others as alpha beta gamma-trimers. The four proteins, of which alpha-subunits were confirmed to be a family of G0-type by an immunoblot analysis, were thus referred to as alpha (0)1, G(0)2, G(0)3 and G(0)4, respectively, in order of their elutions from the column. Immunostained peptide mappings arising from proteolytic digestions of the four alpha-subunits, together with their fragmentation patterns containing radiolabeled ADP-ribose that had been incorporated by pertussis toxin-catalyzed ADP-ribosylation, suggested that the four G0-alpha were classified into either of two groups such as alpha (0)1 and G(0)2-alpha, or G(0)3-alpha and G(0)4-alpha. The kinetic parameters of their GTPase activities, however, revealed that there were different properties between alpha (0)1 and G(0)2-alpha or G(0)3-alpha and G(0)4-alpha. Thus, the four G0-type proteins appeared to be different entities from one another.  相似文献   

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