Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Most genes involved in DNA replication in the yeast Saccharomyces cerevisiae are transcribed transiently during late G1 as cells undergo START. Their promoters all contain one or more versions of an 8-base pair motif (ACGCGTNA) called the MluI cell cycle box (MCB). MCBs have been shown to be both necessary and sufficient for the late G1-specific transcription of the TMP1 thymidylate synthase and POLI DNA polymerase genes. A different late G1-specific transcription element called the SCB (CACGAAAA) is bound by a factor containing the SWI4 and SWI6 proteins. We describe here the formation in vitro of complexes on TMP1 MCBs that contain the SWI6 protein and, we suggest, a 120 kDa protein that is distinct from SWI4. Transcription due to SCBs and MCBs occurs in the absence of SWI6 but it is no longer correctly cell cycle regulated. We suggest that SWI6 is an essential regulatory subunit of two different START-dependent transcription factors. One factor (SBF) contains SWI4 and binds to SCBs whereas the other (MBF) contains p120 and binds MCBs.
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PMID:SWI6 is a regulatory subunit of two different cell cycle START-dependent transcription factors in Saccharomyces cerevisiae. 129 53

We have previously shown that treatment of the HT29 human colorectal tumor (HCT) cell line with 100 nM 5-fluorodeoxyuridine (FdUrd) induces DNA fragments ranging from 50 kilobases to 5 megabases. The studies reported here were conducted to characterize the kinetics, concentration dependence, and pharmacologic specificity of this process and to determine if such fragmentation varies among HCT cell lines. HT29 and SW620 cells yielded similar fragment size distributions upon treatment with either FdUrd or CB3717 [a folate analog inhibitor of thymidylate synthase (TS)]. With either of these agents the SW620 line required higher drug concentrations or longer incubation times than HT29 cells to achieve a given level of fragmentation or cytotoxicity, even though the two cell lines are equally sensitive to FdUrd-induced TS inhibition. These data indicate that SW620 resistance is not due to a lesion in the events leading up to TS inhibition but it may be due to a difference in the steps following TS inhibition. Aphidicolin, a DNA polymerase inhibitor, did not cause substantial fragmentation or cytotoxicity in these two cell lines, demonstrating that the fragmentation response to the other two drugs is not a general consequence of DNA synthesis inhibition. A third HCT line, HuTu80, gave rise only to a smaller and more discrete population of DNA fragments, ranging from approximately 50 to 200 kilobases, following exposure to FdUrd. Similar patterns were seen in this line upon treatment with CB3717 or aphidicolin, indicating that this fragmentation pattern is not specific to TS inhibition and may be characteristic of a more general response than that seen in the other two cell lines. DNA fragments induced by FdUrd in HuTu80 cells did not degrade into smaller pieces, demonstrating that the process by which they are formed is distinct from apoptosis. We conclude that the responses of HCT cells to FdUrd-induced TS inhibition vary significantly, that these differences may reflect heterogeneity in the mechanism of DNA damage formation, and that, in some cases, FdUrd resistance may be due to alterations in the fragmentation process.
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PMID:Variations in patterns of DNA damage induced in human colorectal tumor cells by 5-fluorodeoxyuridine: implications for mechanisms of resistance and cytotoxicity. 143 36

Examples of collateral sensitivity, even in experimental tumor systems, remain few. Preliminary data from this laboratory indicated that certain tumor cells expressed increased sensitivity to cisplatin after exposure in vitro to x-irradiation. To further clarify whether the type of fractionated radiation procedure used clinically can induce hypersensitivities to certain antitumor drugs we have pre-exposed the human ovarian carcinoma cell line JA-T/P derived from a tumor from an untreated patient to fractionated x-irradiation (total dose 50 Gy) in vitro. The resultant subline JA-T/DXR-10 expressed collateral sensitivity to cisplatin (CDDP), methotrexate (MTX) and fluorouracil (5-FU), but not to acute x-irradiation. Hypersensitivity to CDDP was associated with decreased activity of DNA polymerase beta (3.5-fold, P less than .01), but unaltered glutathione metabolism. Pre-incubation with cyclosporin A or with 3-aminobenzamide significantly enhanced (twofold, P less than .01) CDDP-induced cytotoxicity in JA-T/P cells, but not in the DXR-10 subline. Consistent with MTX hypersensitivity dihydrofolate reductase activity was significantly decreased (2.9-fold, P less than .01). Despite collateral sensitivity to 5-FU, however, thymidylate synthase activity was increased (twofold, P less than .05) suggesting alternative mechanisms for 5-FU-induced cytotoxicity in these JA-T/DXR-10 cells. These data demonstrate that DNA repair and associated reduced folate metabolism can be modified not only by drugs but also by fractionated x-irradiation.
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PMID:Expression of collateral sensitivity to cisplatin, methotrexate, and fluorouracil in a human ovarian carcinoma cell line following exposure to fractionated x-irradiation in vitro. 155 59

Most genes involved in DNA replication in the yeast Saccharomyces cerevisiae are transcribed transiently during late G1 as cells become committed to a new cell cycle at Start. Their promoters all contain one or more versions of an 8-base-pair motif (ACGCGTNA) containing an MluI restriction enzyme site and called the MluI cell-cycle box (MCB). MCBs are both necessary and sufficient for the late G1-specific transcription of the TMP1 thymidylate synthase and POL1 DNA polymerase genes. A different late G1-specific 8-base-pair transcription element called the SCB (CACGAAAA; ref. 5) is bound by a factor containing the Swi4 and Swi6 proteins. We describe here the formation in vitro of complexes on TMP1 MCBs that contain the Swi6 protein and, we suggest, a protein of relative molecular mass 120,000 (p120) that is distinct from Swi4. Transcription due to SCBs and MCBs occurs in the absence of Swi6 but it is no longer correctly regulated in the cell cycle. We suggest that Swi6 is an essential regulatory subunit of two different Start-dependent transcription factors. One factor (SBF) contains Swi4 and binds to SCBs, whereas the other (MBF) contains the protein p120 and binds MCBs.
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PMID:A central role for SWI6 in modulating cell cycle Start-specific transcription in yeast. 160 51

The enzymes of DNA polymerization and DNA precursor synthesis are assembled in the replitase complex during the S phase of the cell cycle. Cross-inhibition is a phenomenon shown by enzymes of the replitase complex, in which inhibition of one enzyme of the complex leads to inhibition of a second, unrelated enzyme. This inhibition occurs only in vivo and only during S phase. The second enzyme shows no inhibition in vitro. In this study, using Chinese hamster embryo fibroblast cells, we have shown that direct allosteric interactions, i.e., structural interaction from a remote site within the replitase complex, is the cause of cross-inhibition of thymidylate synthase activity by the inhibitors of ribonucleotide reductase and DNA polymerase, because disruptions of the deoxynucleotide pools, which would be predicted for alternative explantations, do not occur. Cross-inhibition of DNA polymerase by hydroxyurea is demonstrated by the cessation of DNA synthesis when ribonucleotide reductase block is circumvented by the provision of all four deoxynucleosides. In addition to the cross-inhibition for thymidylate synthase and DNA polymerase, we have also presented evidence, on the basis of alterations of the in vivo conversion of deoxyuridine to dUMP, that cross-inhibition also occurs for the enzyme thymidine kinase. This conclusion is further supported by the lack of inhibition of the similar process in RNA synthesis, because enzymes of RNA synthesis are not included in the replitase complex. To facilitate the measurements, we have introduced a novel method of distinguishing between thymidine and deoxyuridine derivatives, making use of the fact that a tritium label placed in the 5'-position of deoxyuridine is removed on conversion to thymidine by methylation, whereas a tritium placed in the 6'-position is not.
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PMID:Allosteric interaction of components of the replitase complex is responsible for enzyme cross-inhibition. 169 15

The intracellular distribution and localization of dihydrofolate reductase-thymidylate synthase of wild type suspension carrot cells was analysed using cytochemical and immunocytochemical techniques; in both resting and growing normal cells (E4) the activity appeared to be predominantly cytoplasmic. The pattern of localization of the enzyme was also analysed during the different phases of the cell cycle. To this end carrot cells were synchronized with aphidicolin (an inhibitor of the alpha-like DNA polymerase which blocks cells at the G1/S boundary) and cycle phases checked by labelled-thymidine incorporation. Protoplasts obtained from cells inhibited with aphidicolin or from cells sampled at different times after the removal of the drug (S and G2 phase), failed to show a nuclear localization of DHFR-TS. These results indicate that in carrot the bifunctional enzyme does not change compartment during the cell cycle. Surprisingly Mtx-resistant cells (E2A2, E2A1C6; overproducing DHFR-TS) showed, irrespective of their physiological state (quiescent or growing), also a relevant nuclear or perinuclear immunofluorescence which could not be detected using cytochemical techniques. The reason of this altered localization is not clear and its possible relation with altered cytophysiological parameters is discussed.
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PMID:Cellular localization of dihydrofolate reductase-thymidylate synthase in carrot cells. 186 64

Enhanced DNA repair has been identified as a major mechanism of resistance to the anticancer drug cisplatin in murine leukemia L1210 cells. Studies of other cells have implicated the elevation of a variety of RNA transcripts in cisplatin resistance. This study investigated potential changes in transcription of these genes as well as genes involved in DNA repair. No elevation in any of the following transcripts was observed: thymidylate synthase, dihydrofolate reductase, DNA polymerase alpha, DNA polymerase beta, topoisomerase II, Ha-ras, beta-tubulin, metallothionein and the DNA repair genes ERCC1 and ERCC2. Thymidine kinase was increased no more than 2-fold. None of these RNA were induced by incubation with cisplatin. High levels of cisplatin produced selective decreases in certain RNA. These results demonstrate that the previous observations of elevated RNA can not be universally applied to all cisplatin-resistant cells.
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PMID:Analysis of various mRNA potentially involved in cisplatin resistance of murine leukemia L1210 cells. 197 66

The DNA polymerase alpha inhibitor, aphidicolin, was employed to synchronize large-scale suspension cultures (10(9) cells) of murine L1210 leukemia cells. On the basis of the doubling time and cell cycle distribution for logarithmically growing L1210 cells, a synchronization protocol was devised involving a temporal sequence of two 12-h exposures to aphidicolin, separated by an 6-h interval in drug-free medium. After the second aphidicolin treatment, resuspension of cells into drug-free medium resulted in the rapid onset of DNA synthesis as assessed by [3H]thymidine incorporation and DNA fluorescence with flow cytometry. By 6 h after aphidicolin removal, the cells progressed into the G2-M phase and cell division was initiated. DNA synthesis was minimal during this time and remained low through 9 h when the majority of the cells were in G1 phase. Only low levels of cytotoxicity were observed when L1210 cells were treated with aphidicolin in this fashion. The levels of both thymidylate synthase and dihydrofolate reductase were relatively constant during cell cycle transit, following release from the aphidicolin blockade. Similarly, the levels of the corresponding mRNA transcripts for these enzymes, measured by Northern blot hybridizations, remained essentially unchanged through most of the cell cycle, increasing approximately twofold only as the cells entered G1 phase. Whereas intracellular dihydrofolate reductase catalytic activity was relatively unchanged throughout the cell cycle, as reflected in the metabolism of [3H]folic acid to reduced folate forms, a marked increase in in situ thymidylate synthase activity occurred during S phase that was tightly linked to the rate of DNA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A method for the synchronization of cultured cells with aphidicolin: application to the large-scale synchronization of L1210 cells and the study of the cell cycle regulation of thymidylate synthase and dihydrofolate reductase. 251 11

Exponentially growing human lymphoblasts (culture LS-2) were separated by cell sorting (FACS II, Becton Dickinson) according to their deoxyribonucleic acid (DNA) content, designating them at particular phases of the cell cycle. Prior to cell sorting the DNA has been fluorochrome-labeled with the Hoechst stain H 33342. Maximum cell enrichments of 94% for G0 + G1 cells, 96% for S cells and 74% for G2 + M cells could be achieved. The enzyme activities of thymidine kinase (TK), thymidylate synthase (TS), DNA polymerase (DNA-P), dihydrofolate reductase (FH2-R), methionine synthase (MS), and hexokinase (HK) were determined in the obtained cell fractions. Although incorporation of 3H-thymidine (3H-dTR) and the 3H-dTR labeling index were significantly inhibited by the dye, no evidence of cell staining's having a significant effect on the enzyme activities was found. The enzyme activities for approximately 100% pure G0 + G1, S, and G2 + M cells were computed. With exception of TK, all the enzymes under study were shown to exhibit activities--although of differing degree--in the G0 + G1, S, and G2 + M cells. No TK activity was shown in G0 and G1 cells; its activity, however, was approximately the same in S and G2 + M cells. This applies likewise for TS which, in contrast to TK, exhibits minor activity in G0 + G1 cells. DNA-P was highly active in G0 + G1 cells, but maximum activity was in S cells. FH2-R exhibited maximum activity in S cells, although the difference in activity between S and G2 + M cells was not significant. None of the observed differences in MS activity was significant, indicating equally high activity in cells of all cell cycle phases. HK activity is approximately twice as high in G2 + M cells as in G0 + G1 cells.
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PMID:Relation between cell cycle stage and the activity of DNA-synthesizing enzymes in cultured human lymphoblasts: investigations on cells separated according to DNA content by way of a cell sorter. 271 50

Undifferentiated human lymphoblasts (culture LS-2) were separated according to cell size during their exponential growth phase by way of centrifugal elutriation. The cell fractions thus obtained were characterized in terms of different cell cycle stages by flow cytometric measurement of their deoxyribonucleic acid (DNA histogram), the [3H]thymidine labeling index, and by determining the rate of [3H]thymidine incorporation. In these cell fractions the activities of thymidine kinase, thymidylate synthase, DNA polymerase, dihydrofolate reductase, methionine synthase, and hexokinase were determined. The results showed that all the enzymes investigated exhibited activities in all cell fractions. With the exception of DNA polymerase, all of the enzymes exhibited the lowest level of activity in the fraction containing the highest proportion of G0 + G1 phase cells (fraction 2); the activity of thymidine kinase was particularly low. This would suggest that thymidine kinase is not active in G0 + G1 phase cells and that the activity measured in fraction 2 is perhaps attributable to contamination of this fraction by S and G2 + M phase cells.
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PMID:Relation between cell cycle stage and the activity of DNA-synthesizing enzymes in cultured human lymphoblasts: investigations on cell fractions enriched according to cell cycle stages by way of centrifugal elutriation. 366 41


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