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)

Although a number of transfection experiments have suggested potential targets for the action of the E2F1 transcription factor, as is the case for many transcriptional regulatory proteins, the actual targets in their normal chromosomal environment have not been demonstrated. We have made use of a recombinant adenovirus containing the E2F1 cDNA to infect quiescent cells and then measure the activation of endogenous cellular genes as a consequence of E2F1 production. We find that many of the genes encoding S-phase-acting proteins previously suspected to be E2F targets, including DNA polymerase alpha, thymidylate synthase, proliferating cell nuclear antigen, and ribonucleotide reductase, are indeed induced by E2F1. Several other candidates, including the dihydrofolate reductase and thymidine kinase genes, were only minimally induced by E2F1. In addition to the S-phase genes, we also find that several genes believed to play regulatory roles in cell cycle progression, such as the cdc2, cyclin A, and B-myb genes, are also induced by E2F1. Moreover, the cyclin E gene is strongly induced by E2F1, thus defining an autoregulatory circuit since cyclin E-dependent kinase activity can stimulate E2F1 transcription, likely through the phosphorylation and inactivation of Rb and Rb family members. Finally, we also demonstrate that a G1 arrest brought about by gamma irradiation is overcome by the overexpression of E2F1 and that this coincides with the enhanced activation of key target genes, including the cyclin A and cyclin E genes.
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PMID:Cellular targets for activation by the E2F1 transcription factor include DNA synthesis- and G1/S-regulatory genes. 762 16

A variety of studies have now implicated the cellular transcription factor E2F as a key participant in transcription control during the cell growth cycle. Although the recent isolation of molecular clones encoding proteins that are components of the E2F activity (E2F1 and DP-1) provides an approach to defining the specific involvement of E2F in these events, definitive experiments remain difficult in the absence of appropriate genetic systems. We have now identified a Drosophila equivalent of E2F1 that we hope will allow an eventual genetic approach to the role of E2F in cellular regulatory events. A cDNA clone was isolated from a Drosophila cDNA library by using a probe containing sequence from the E2F1 DNA binding domain. The sequence of the clone, which we term drosE2F1, demonstrates considerable homology to the human E2F1 sequence, with over 65% identity in the DNA binding region and 50% identity in the region of E2F1 known to interact with the retinoblastoma gene product. A glutathione S-transferase-drosE2F1 fusion protein was capable of binding specifically to an E2F recognition site, and transfection assays demonstrated that the drosE2F1 product was capable of transcription activation, dependent on functional E2F sites as well as sequences within the C terminus of the protein. Finally, we have also identified E2F recognition sequences within the promoter of the Drosophila DNA polymerase alpha gene, and we demonstrate that the drosE2F1 product activates transcription of a test gene under the control of this promoter. We conclude that the drosE2F1 cDNA encodes an activity with extensive structural and functional similarity to the human E2F1 protein.
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PMID:Functional properties of a Drosophila homolog of the E2F1 gene. 811 98

E2F has been implicated in growth control because of its association with the retinoblastoma protein and the presence of E2F binding sites in the promoters of several growth-regulated genes. Proteins that bind to an E2F site have been cloned from human and mouse cells. However, these two proteins (human E2F1 and mouse DP-1) are quite different in sequence. We have now cloned a mouse cDNA encoding a protein 86% identical to the human E2F1 protein. The mouse E2F1 cDNA encodes a 430-amino-acid protein with a predicted molecular weight of 46,322 and detects mRNAs of 2.7 and 2.2 kb. Using primers complementary to sequences in the mouse E2F1 3' untranslated region, we mapped the mouse E2F1 gene to chromosome 2, near the Agouti and c-src loci. To understand the role of the different E2F family members in the growth of mouse NIH 3T3 cells, we examined the levels of E2F1 and DP-1 mRNAs in different stages of the cell cycle. Since the levels of E2F1 but not DP-1 mRNA correlated with changes in transcription from the dhfr promoter, we examined whether E2F1 could activate various growth-regulated promoters. We found that E2F1 could activate some (dhfr, thymidine kinase, and DNA polymerase alpha) but not all (thymidylate synthase, cad, and c-myc) of these promoters. On the basis of changes in levels of E2F1 and its ability to transactivate growth-regulated promoters, we propose that E2F1 may mediate growth factor-initiated signal transduction.
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PMID:Cloning, chromosomal location, and characterization of mouse E2F1. 811 19

The amino terminus of the E2F1 transcription factor is a protein-protein interaction domain since it associates with cyclin A/cdk2. Here, the two-hybrid yeast screen was used to clone genes whose products associate with the amino terminus of E2F1. The amino-terminal 121 amino acids of E2F1 were fused to the Lex A binding domain while a partial length cDNA library from the embryo of a 12 day old mouse was fused to the VP16 activation domain. Following coexpression of these fusions in yeast, two novel genes were cloned that code for proteins that associate with E2F1. In an in vitro assay, these E2F1 Binding Proteins (EBP1 and EBP2) associate with residues 1-121 of E2F1 or with the full-length protein; however, they do not associate with its carboxy terminus (residues 88-437). When EBP1 or EBP2 were expressed in COS cells along with E2F1 and the target promoter DNA polymerase alpha, repression of transcription was observed. However, no repression of DNA polymerase alpha was seen if the cells expressed a nonassociating mutant E2F1 (residues 88-437), along with EBP1 or EBP2. Finally, expression of the EBP2 gene is up-regulated in growing NIH3T3 fibroblasts, relative to serum-starved cells. However, this up-regulation of EBP2 expression is not seen in fibroblasts constitutively expressing E2F1.
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PMID:Isolation of two novel cDNAs whose products associate with the amino terminus of the E2F1 transcription factor. 882 66

The products of the tumor suppressor genes are considered to function as specific inhibitors of tumor cell growth. In this communication, we present evidence to show that these proteins inhibit tumor cell proliferation by participating in the activation of tumor cell differentiation. The ML-1 human myeloblastic leukemia cells used in this study proliferate when treated with insulin-like growth factor I and transferrin but differentiate to monocytes when exposed to tumor necrosis factor alpha or transforming growth factor beta1, or to macrophage-like cells when treated with both these cytokines. Initiation of proliferation but not of differentiation was followed by a 20- to 25-fold increase in the nuclear level of the DNA polymerase-associated processivity factor PCNA and of the proliferation-specific transcription factor E2F1. In contrast, induction of differentiation but not of proliferation was followed by a 25- to 30-fold increase in the nuclear level of the tumor suppressor proteins p53 (wild type), pRb, and p130/Rb2 and of the p53-dependent cyclin kinase inhibitor p21/Cip1. p53 and p21/Cip1, respectively, inhibit the expression and activation of PCNA, whereas p130 and pRb, respectively, inhibit the expression and activation of E2F1. As a result, G1-S-associated DNA and mRNA synthesis is inhibited, growth uncoupled from differentiation, and maturation enabled to proceed. Where this function of the tumor suppressor proteins is impaired, the capacity for differentiation is lost, which leads to the sustained proliferation that is characteristic of the cancer cell.
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PMID:Tumor suppressor proteins as regulators of cell differentiation. 976 53

The Epstein-Barr Virus (EBV) immediate-early protein BRLF1 is one of two transactivators which mediate the switch from latent to lytic replication in EBV-infected cells. DNA viruses often modulate the function of critical cell cycle proteins to maximize the efficiency of virus replication. Here we have examined the effect of BRLF1 on cell cycle progression. A replication-deficient adenovirus expressing BRLF1 (AdBRLF1) was used to infect normal human fibroblasts and various epithelial cell lines. BRLF1 expression induced S phase entry in contact-inhibited fibroblasts and in the human osteosarcoma cell line U-2 OS. AdBRLF1 infection produced a dramatic increase in the level of E2F1 but not E2F4. In contrast, the levels of Rb, p107, and p130 were decreased in AdBRLF1-infected cells. Electrophoretic mobility shift assays confirmed an increased level of free E2F1 in the AdBRLF1-infected human fibroblasts. Consistent with the previously described effect of E2F1, AdBRLF1-infected fibroblasts had increased levels of p53 and p21 and died by apoptosis. BRLF1-induced activation of E2F1 may be required for efficient EBV lytic replication, since at least one critical viral replication gene (the viral DNA polymerase) is activated by E2F (C. Liu, N. D. Sista, and J. S. Pagano, J. Virol. 70:2545-2555, 1996).
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PMID:The Epstein-Barr virus protein BRLF1 activates S phase entry through E2F1 induction. 1040 Jul 50

The B-subunits of replicative DNA polymerases from Archaea to humans belong to the same protein family, suggesting that they share a common fundamental function. We report here the gene structure for the B-subunit of human DNA polymerase epsilon (POLE2), whose expression and transcriptional regulation is typical for replication proteins with some unique features. The 75 bp core promoter region, located within exon 1, contains an Sp1 element that is a critical determinant of promoter activity as shown by the luciferase reporter, electrophoretic mobility shift and DNase I footprinting assays. Two overlapping E2F elements adjacent to the Sp1 element are essential for full promoter activity and serum response. Binding sites for E2F1 and NF-1 reside immediately downstream from the core promoter region. Our results suggest that human POLE2 is regulated by two E2F-pocket protein complexes, one associated with Sp1 and the other with NF-1. So far, only one replicative DNA polymerase B-subunit gene promoter, POLA2 encoding the B-subunit of DNA polymerase alpha, has been characterized. Mitogenic activation of the POLE2 promoter by an E2F-mediated mechanism resembles that of POLA2, but the regulation of basal promoter activity is different between these two genes.
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PMID:E2F mediates induction of the Sp1-controlled promoter of the human DNA polymerase epsilon B-subunit gene POLE2. 1143 27

Various studies have demonstrated a role for E2F proteins in the control of transcription of genes involved in DNA replication, cell cycle progression, and cell fate determination. Although it is clear that the functions of the E2F proteins overlap, there is also evidence for specific roles for individual E2F proteins in the control of apoptosis and cell proliferation. Investigating protein interactions that might provide a mechanistic basis for the specificity of E2F function, we identified the E-box binding factor TFE3 as an E2F3-specific partner. We also show that this interaction is dependent on the marked box domain of E2F3. We provide evidence for a role for TFE3 in the synergistic activation of the p68 subunit gene of DNA polymerase alpha together with E2F3, again dependent on the E2F3 marked box domain. Chromatin immunoprecipitation assays showed that TFE3 and E2F3 were bound to the p68 promoter in vivo and that the interaction of either E2F3 or TFE3 with the promoter was facilitated by the presence of both proteins. In contrast, neither E2F1 nor E2F2 interacted with the p68 promoter under these conditions. We propose that the physical interaction of TFE3 and E2F3 facilitates transcriptional activation of the p68 gene and provides strong evidence for the specificity of E2F function.
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PMID:Identification of E-box factor TFE3 as a functional partner for the E2F3 transcription factor. 1274 76

MCM10 and TopBP1 function in the initiation of DNA replication, by regulating the chromatin binding of the DNA polymerase alpha loading factor, CDC45. TopBP1 is also known as a DNA damage response protein. In this study, we showed that the transcription of human MCM10 and TopBP1 is activated by transcription factors E2F1-3, but not by factors E2F4-7. Analysis of various MCM10 and TopBP1 promoter constructs showed that an E2F-responsive sequence in the vicinity of the transcription initiation site is necessary for the E2F1-induced activation of MCM10 and TopBP1 gene transcription, which is further suppressed by pRb. The promoter activities of human MCM10 and TopBP1 were demonstrated to be growth dependent via the E2F-responsive sequence. Although E2F1 was stabilized by ultraviolet (UV) irradiation, the mRNA expression level of TopBP1 was suppressed in HCT116 human diploid colon cancer cells. We showed, by performing chromatin immunoprecipitation that, in response to UV irradiation but not doxorubicin treatment, E2F4 accumulated on the MCM10 and TopBP1 promoters. Our data suggest a model in which UV irradiation-induced DNA damage depends, at least in part, on the accumulation of the E2F4 transcription factor on the MCM10 and TopBP1 promoters, which results in suppression of DNA replication.
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PMID:Expression of MCM10 and TopBP1 is regulated by cell proliferation and UV irradiation via the E2F transcription factor. 1519 43

Pole3 (DPB4/YBL1/CHRAC17) is one of the subunits of the DNA polymerase e. It contains a histone-like domain required for the hererodimerization with its Pole4 (DPB3) partner. In another interaction, Pole3 heterodimerizes with YCL1/CHRAC15 and associates with the ACF1/SNF2H remodelling complex. We find that the Pol3 gene is regulated in starved NIH3T3 fibroblasts upon induction with serum, with a peak at the entry in the S phase. We characterized the Pole3 promoter, which is linked bidirectionally to C9Orf46, a gene of unknown function: it has no CCAAT nor TATA-boxes, and contains an E box and two potential E2F sites. Mutagenesis analysis points to a minimal promoter region as sufficient for activation; the E box and a neighbouring direct repeat are important for regulation. Cell-cycle regulation was reproduced in stable clones and an additional E2F site was found to be important. Chromatin immunoprecipitation analysis indicates that E2F1/4, as well as MYC, are associated with the Pole3 promoter in a phase-specific way. These data highlight coregulation of a histone-like gene with core histones upon DNA synthesis, and represent a first dissection of the interplay between two essential cell-cycle regulators on a bidirectional promoter.
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PMID:The Pole3 bidirectional unit is regulated by MYC and E2Fs. 1640 26


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