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)

Human retinoblastoma (Rb) protein, immunopurified from an extract of recombinant baculovirus infected cells, stimulated 10-100-fold the activity of DNA polymerase alpha from calf thymus or human HeLa cells. Purified Rb protein is composed of two electrophoretically distinguishable forms, i.e., partially phosphorylated and under-phosphorylated forms. Dephosphorylation of Rb protein by protein phosphatase 2A largely diminished its stimulatory effect. On the other hand, a hyperphosphorylated Rb protein, obtained from insect cells overexpressing Rb protein, cyclin E and cyclin-dependent kinase 2 simultaneously, stimulated DNA polymerase alpha more strongly than the singly-expressed Rb protein. These results indicate that the phosphorylation is crucial for the stimulation. Rb protein isolated from human Burkitt lymphoma Raji cells also stimulated DNA polymerase alpha. In contrast, Rb protein did not affect eukaryotic DNA primase or Klenow fragment of Escherichia coli DNA polymerase I. By immunoprecipitation using anti-DNA polymerase alpha antibody, Rb protein in nuclear extract of Raji cells was co-precipitated with DNA polymerase alpha. This result indicates that DNA polymerase alpha exists as a complex containing phosphorylated Rb protein in cells. DNA polymerase alpha specifically bound to a purified Rb protein-immobilized Sepharose column. Rb protein also bound to DNA polymerase alpha trapped to anti-DNA polymerase alpha antibody-Sepharose column, suggesting the direct association of these two proteins. These observations suggest a new function of phosphorylated Rb protein in the regulation of DNA replication.
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PMID:Phosphorylated retinoblastoma protein stimulates DNA polymerase alpha. 939 44

Phosphorylated retinoblastoma protein and nucleolar protein B23 are putative stimulatory factors for DNA polymerase alpha. We showed that these two factors interacted with each other and stimulated the activity of DNA polymerase alpha synergistically. B23 exists in two isoforms designated as B23.1 and B23.2. While B23.1 bound to a retinoblastoma protein-conjugated column, B23.2 did not. These results indicate that B23.1 can directly bind to retinoblastoma protein. It was also shown that B23 was co-immunoprecipitated with both retinoblastoma protein and DNA polymerase alpha from a HeLa cell extract by monoclonal antibodies raised against these components. These results suggest that these three proteins exist as a complex in cells, at least in part. The simultaneous addition of both B23.1 and retinoblastoma protein caused stimulation of DNA polymerase alpha activity that is much higher than the sum of the stimulation by retinoblastoma protein and B23.1 alone. The maximal stimulation was attained at the molar ratio of DNA polymerase alpha/retinoblastoma protein/B23.1 = 1:1:12. Since B23 exists as a hexamer in solution, it may act as a stimulator of DNA polymerase alpha in a form of double-hexamer, in concert with the phosphorylated retinoblastoma protein.
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PMID:Nucleolar protein B23.1 binds to retinoblastoma protein and synergistically stimulates DNA polymerase alpha activity. 1022 May 82

We propose a new role of retinoblastoma protein as a cell growth activator in its phosphorylated form. The hyper-phosphorylated retinoblastoma protein generated by the action of cdk2/cyclin E strongly stimulated the activity of DNA polymerase alpha, but did not stimulate DNA polymerases delta, epsilon, or primase. But, cdk4/cyclin D-phosphorylated retinoblastoma protein showed little stimulation. Hyper-phosphorylated retinoblastoma protein interacted with the catalytic subunit of DNA polymerase alpha, and stabilised DNA polymerase alpha from heat inactivation at 45 degrees C. These results suggest that in G1 phase, hypo-phosphorylated retinoblastoma protein suppresses the progression of cell cycle as a transcription inhibitor, but that after phosphorylation by cdk2/cyclin E at the G1/S boundary, hyper-phosphorylated retinoblastoma protein acts as a cell-cycle promoter by optimising the DNA polymerase alpha reaction.
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PMID:Stimulation of DNA polymerase alpha activity by cdk2-phosphorylated Rb protein. 1135 49

The balance between cell differentiation and proliferation is regulated at the transcriptional level. In the cell cycle, the transition from G1 to S phase (G1/S transition) is of paramount importance in this regard. Indeed, it is only before this point that cells can be oriented toward the differentiation pathway: beyond, cells progress into the cycle in an autonomous manner. The G1/S transition is orchestrated by the transcription factor E2F. E2F controls the expression of a group of checkpoint genes whose products are required either for the G1-to-S transition itself or for DNA replication (e.g. DNA polymerase alpha). E2F activity is repressed in growth-arrested cells and in early G1, and is activated at mid-to-late G1. E2F is controlled by the retinoblastoma tumor suppressor protein Rb. Rb represses E2F mainly by recruiting chromatin remodeling factors (histone deacetylases and SWI/SNF complexes), the DNA methyltransferase DNMT1, and a histone methyltransferase. This review will focus on the molecular mechanisms of E2F repression by Rb during the cell cycle and during cell-cycle exit by differentiating cells. A model in which Rb irreversibly represses E2F-regulated genes in differentiated cells by an epigenetic mechanism linked to heterochromatin, and involving histone H3 and promoter DNA methylation, is discussed.
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PMID:The Rb/chromatin connection and epigenetic control: opinion. 1142 Jul 29

Combinatorial fluorescence energy transfer (CFET) tags, constructed by exploiting energy transfer and combinatorial synthesis, allow multiple biological targets to be analyzed simultaneously. We here describe a multiplex single nucleotide polymorphism (SNP) assay based on single base extension (SBE) using CFET tags and biotinylated dideoxynucleotides (biotin-ddNTPs). A library of CFET-labeled oligonucleotide primers was mixed with biotin-ddNTPs, DNA polymerase and the DNA templates containing the SNPs in a single tube. The nucleotide at the 3'-end of each CFET-labeled oligonucleotide primer was complementary to a particular SNP in the template. Only the CFET-labeled primer that is fully complementary to the DNA template was extended by DNA polymerase with a biotin-ddNTP. We isolated the DNA extension fragments that carry a biotin at the 3'-end by capture with streptavidin-coated magnetic beads, while the unextended primers were eliminated. The biotinylated fluorescent DNA fragments were subsequently analyzed in a multicolor fluorescence electrophoresis system. The distinct fluorescence signature and electrophoretic mobility of each DNA extension product in the electropherogram coded the SNPs without the use of a sizing standard. We simultaneously distinguished six nucleotide variations in synthetic DNA templates and a PCR product from the retinoblastoma tumor suppressor gene. The use of CFET-labeled primers and biotin-ddNTPs coupled with the specificity of DNA polymerase in SBE offered a multiplex method for detecting SNPs.
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PMID:Single nucleotide polymorphism detection by combinatorial fluorescence energy transfer tags and biotinylated dideoxynucleotides. 1186 24

Previously, my colleagues and I have reported that the immunopurified hyper-phosphorylated retinoblastoma protein (ppRb) stimulates the activity of DNA polymerase alpha. I describe here the biochemical characteristics of this stimulatory activity. DNA polymerase alpha-stimulatory activity of ppRb was most remarkable when using activated DNA as a template-primer, rather than using poly(dT)-(rA)(10), poly(dA)-(dT)(12-18), and so on. Kinetic analysis showed that there was no significant difference in K(m) value for deoxyribonucleotides of DNA polymerase alpha in the presence of ppRb. Adding ppRb resulted in the overcoming pause site on the template, but did not affect the rate of misincorporation of incorrect deoxyribonucleotides. By adding ppRb, the optimal concentration of template-primer was shifted to a higher region, but not using M13 singly primed DNA. The ppRb seemed to assist the process that DNA polymerase alpha changed its conformation resulting in appropriate enzyme activity. These results suggest that ppRb affects both template-primer and DNA polymerase alpha and makes appropriate circumstances for the enzyme reaction.
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PMID:Biochemical properties of the stimulatory activity of DNA polymerase alpha by the hyper-phosphorylated retinoblastoma protein. 1204 95

Not all carcinogens are mutagens, and many mutagens are not carcinogens. Among related chemicals, small changes of structure can markedly influence carcinogenic potency. Many tumours are genetically unstable, but some, especially 'benign' types, rarely exhibit 'progression' or show other evidence of genetic instability. Cells of particular tumour types exhibit identifiable particular 'sets' of phenotypic abnormalities (e.g. rapid growth, uniform nuclei, little cytoplasm and occasionally production of adrenocorticotrophic hormone by anaplastic small-celled carcinoma of the bronchus). Tumour cells pass their abnormalities on to their daughter cells, indicating that a genomic alteration probably underlies tumour formation. A possible mechanism, which might explain these phenomena is carcinogen-induced reduction of fidelity of replication of DNA polymerase complexes during S phase of normal tissue stem cells. A single 'hit' by a reactive agent (chemical or physical) on one of the major enzymic sites (synthesis, proofreading, mismatch repair-MMR) could cause multiple sequence abnormalities in the length of DNA synthesized by one DNA polymerase complex. Because this length of DNA (half a replication 'bubble') averages 15 000-150 000 nucleotides, the affected DNA could include two or more significant genomic elements (genes, especially for tumour suppression, regulatory loci and other elements). The particular mutant elements in the affected DNA could then determine the 'set' of phenotypic abnormalities exhibited by a resulting tumour. Non-genotoxic carcinogenicity, non-carcinogenic mutagenicity, structure-dependent chemical carcinogenicity and the phenomenon of 'sets' of phenotypic abnormalities could thus be accommodated. In experimental studies, the 'hallmark pattern' of mutation caused by this mechanism would be multiple mainly point mutations clustered within the length of half a replication 'bubble'. Such a 'hallmark pattern' of mutation might be detectable in carcinogen-treated cell cultures by the use of cycle-synchronized cultures, single cell subculturing, restriction (endonuclease) fragment length analysis of the clones and nucleotide sequencing of abnormal bands for localization in the human genome. If the mechanism is important to carcinogenesis generally, then non-carcinogenic mutagens should not cause the 'hallmark pattern' of mutations in either in vitro or in vivo systems. In human tumour cells, the 'hallmark pattern' of mutations may be demonstrable in genetically stable human tumours, but might well be lost or obscured by secondary mutations in genetically unstable tumours. Among different cases of the same type of human tumour, the clustered point mutations might be tumour-type specific in their location in the genome, but vary case-to-case in the precise 'points' mutated in the cluster region. New assays for assessing the carcinogenic potential of environmental and synthetic substances for human and animal populations may result. The hypothesis is not put forward to the exclusion of some established mechanisms of carcinogenesis for particular human tumours: for example, the 'two-hit' mutational hypothesis for retinoblastoma, the 'multiple sequential mutational' hypothesis for UV-induced lesions of the epidermis, and the possibility of adduct-induced frameshift mutations by some chemical carcinogens for experimental tumours.
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PMID:Carcinogen-induced impairment of enzymes for replicative fidelity of DNA and the initiation of tumours. 1460 90

p12(CDK2-AP1) (p12) is a growth suppressor isolated from normal keratinocytes. Ectopic expression of p12 in squamous carcinoma cells reversed the malignant phenotype of these cells, in part due an ability of p12 to bind to both DNA polymerase alpha/primase and to cyclin-dependent kinase 2 (CDK2), thereby inhibiting their activities. We report in this article that in normal epithelial cells, transforming growth factor beta1 (TGF-beta1) induces p12 expression transcriptionally, which, in turn, mediates the growth inhibitory activity of TGF-beta1. We created inducible p12 antisense HaCaT cell lines [ip12 (-) HaCaT] and showed that selective reduction of cellular p12 resulted in an increase in: (a) CDK2-associated kinase activity; (b) protein retinoblastoma (pRB) phosphorylation; and (c) [(3)H]thymidine incorporation, and partially reversed TGF-beta1-mediated inhibition of CDK2 kinase activity, pRB phosphorylation, and cell proliferation. Furthermore, we generated p12-deficient mouse oral keratinocytes (MOK(p12-/-)) and compared their growth characteristics and response to TGF-beta1 with that of wild-type mouse oral keratinocytes (MOK(WT)). Under normal culture conditions, the number of MOK(p12-/-) in S phase is 2-fold greater than that of MOK(WT). Concomitantly, fewer cells are in G(2) phase in MOK(p12-/-) than that in MOK(WT). Moreover, response to TGF-beta1-mediated growth suppression is compromised in MOK(p12-/-) cells. Mechanistic studies showed that MOK(p12-/-) have increased CDK2 activity and reduced sensitivity to inhibition by TGF-beta1. Collectively our data suggest that p12 plays a role in TGF-beta1-mediated growth suppression by modulating CDK2 activities and pRB phosphorylation.
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PMID:Role of p12(CDK2-AP1) in transforming growth factor-beta1-mediated growth suppression. 1474 61

In vertebrates, MCM2-7 and Cdc45 are required for DNA replication initiation, but it is unknown whether they are also required for elongation, as in yeast. Moreover, although MCM2-7 is a prime candidate for the eukaryotic replicative DNA helicase, a demonstration that MCM2-7 unwinds DNA during replication is lacking. Here, we use Xenopus egg extracts to investigate the roles of MCM7 and Cdc45 in DNA replication. A fragment of the retinoblastoma protein, Rb(1-400), was used to neutralize MCM7, and antibodies were used to neutralize Cdc45. When added immediately after origin unwinding, or after significant DNA synthesis, both inhibitors blocked further DNA replication, indicating that MCM7 and Cdc45 are required throughout replication elongation in vertebrates. We next exploited the fact that inhibition of DNA polymerase by aphidicolin causes extensive chromosome unwinding, likely due to uncoupling of the replicative DNA helicase. Strikingly, Rb(1-400) and Cdc45 antibodies both abolished unwinding by the uncoupled helicase. These results provide new support for the model that MCM2-7 is the replicative DNA helicase, and they indicate that Cdc45 functions as a helicase co-factor.
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PMID:A requirement for MCM7 and Cdc45 in chromosome unwinding during eukaryotic DNA replication. 1532 70

Methionine deprivation imposes a metabolic stress, termed methionine stress, that inhibits mitosis and induces cell cycle arrest and apoptosis. The methionine-dependent central nervous system tumor cell lines DAOY (medulloblastoma), SWB61 (anaplastic oligodendroglioma), SWB40 (anaplastic astrocytoma), and SWB39 (glioblastoma multiforme) were compared with methionine-stress resistant SWB77 (glioblastoma multiforme). The cDNA-oligoarray analysis and reverse transcription-PCR verification indicated common changes in gene expression in methionine-dependent cell lines to include up-regulation/induction of cyclin D1, mitotic arrest deficient (MAD)1, p21, growth arrest and DNA-damage-inducible (GADD)45 alpha, GADD45 gamma, GADD34, breast cancer (BRCA)1, 14-3-3sigma, B-cell CLL/lymphoma (BCL)1, transforming growth factor (TGF)-beta, TGF-beta-induced early response (TIEG), SMAD5, SMAD7, SMAD2, insulin-like growth factor binding protein (IGFBP7), IGF-R2, vascular endothelial growth factor (VEGF), TNF-related apoptosis-inducing ligand (TRAIL), TNF-alpha converting enzyme (TACE), TRAIL receptor (TRAIL-R)2, TNFR-related death receptor (DR)6, TRAF interacting protein (I-TRAF), IL-6, MDA7, IL-1B convertase (ICE)-gamma, delta and epsilon, IRF1, IRF5, IRF7, interferon (IFN)-gamma and receptor components, ISG15, p65-NF-kappaB, JUN-B, positive cofactor (PC)4, C/ERB-beta, inositol triphosphate receptor I, and methionine adenosyltransferase II. On the other hand, cyclins A1, A2, B1 and B2, cell division cycle (CDC)2 and its kinase, CDC25 A and B, budding uninhibited by benzimidazoles (BUB)1 and 3, MAD2, CDC28 protein kinase (CKS)1 and 2, neuroepithelial cell transforming gene (NET)1, activator of S-phase kinase (ASK), CDC14B phosphatase, BCL2, TGF-beta activated kinase (TAK)1, TAB1, c-FOS, DNA topoisomerase II, DNA polymerase alpha, dihydrofolate reductase, thymidine kinase, stathmin, and MAP4 were down-regulated. In the methionine stress-resistant SWB77, only 20% of the above genes were affected, and then only to a lesser extent. In addition, some of the changes observed in SWB77 were opposite to those seen in methionine-dependent tumors, including expression of p21, TRAIL-R2, and TIEG. Despite similarities, differences between methionine-dependent tumors were substantial, especially in regard to regulation of cytokine expression. Western blot analysis confirmed that methionine stress caused the following: (a) a marked increase of GADD45alpha and gamma in the wt-p53 cell lines SWB61 and 40; (b) an increase in GADD34 and p21 protein in all of the methionine-dependent lines; and (c) the induction of MDA7 and phospho-p38 in DAOY and SWB39, consistent with marked transcriptional activation of the former under methionine stress. It was additionally shown that methionine stress down-regulated the highly active phosphatidylinositol 3'-kinase pathway by reducing AKT phosphorylation, especially in DAOY and SWB77, and also reduced the levels of retinoblastoma (Rb) and pRb (P-ser780, P-ser795, and P-ser807/811), resulting in a shift in favor of unphosphorylated species in all of the methionine-dependent lines. Immunohistochemical analysis showed marked inhibition of nuclear translocation of nuclear factor kappaB under methionine stress in methionine-dependent lines. In this study we show for the first time that methionine stress mobilizes several defined cell cycle checkpoints and proapoptotic pathways while coordinately inhibiting prosurvival mechanisms in central nervous system tumors. It is clear that methionine stress-induced cytotoxicity is not restricted by the p53 mutational status.
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PMID:Modulation of gene expression in human central nervous system tumors under methionine deprivation-induced stress. 1549 78


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