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

---

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

The properties of the methyl-directed DNA (cytosine-5-)-methyltransferase (EC 2.1.1.37) suggest that it is the enzyme that maintains patterns of methylation in the human genome. Proposals for the enzyme's mechanism of action suggest that 5-methyldeoxycytidine is produced from deoxycytidine via a dihydrocytosine intermediate. We have used an oligodeoxynucleotide containing 5-fluorodeoxycytidine as a suicide substrate to capture the enzyme and the dihydrocytosine intermediate. Gel retardation experiments demonstrate the formation of the expected covalent complex between duplex DNA containing 5-fluorodeoxycytidine and the human enzyme. Formation of the complex was dependent upon the presence of the methyl donor S-adenosylmethionine, suggesting that it comprises an enzyme-linked 5-substituted dihydrocytosine moiety in DNA. Dihydrocytosine derivatives are extremely labile toward hydrolytic deamination in aqueous solution. Because C-to-T transition mutations are especially prevalent at CG sites in human DNA, we have used high-performance liquid chromatography to search for thymidine that might be generated by hydrolysis during the methyl transfer reaction. Despite the potential for deamination inherent in the formation of the intermediate, the methyltransferase did not produce detectable amounts of thymidine. The data suggest that the ability of the human methyltransferase to preserve genetic information when copying a methylation pattern (i.e., its fidelity) is comparable to the ability of a mammalian DNA polymerase to preserve genetic information when copying a DNA sequence. Thus the high frequency of C-to-T transitions at CG sites in human DNA does not appear to be due to the normal enzymatic maintenance of methylation patterns.
...
PMID:Mechanism of human methyl-directed DNA methyltransferase and the fidelity of cytosine methylation. 158 13

As a step toward the molecular elucidation of the putative replicational apparatus associated with the nuclear matrix, we have investigated the possible matrix association of several replicational related enzymes. In addition to the previously identified DNA polymerase alpha, DNA primase, 3'-5' exonuclease, RNase H, and DNA methylase were all recovered at significant levels (20-30% of total nuclear activity) in nuclear matrix isolated from regenerating rat liver during maximal in vivo replication (22 h post-hepatectomy). In contrast, DNA ligase was not detected on the nuclear matrix even though significant activity was present in isolated nuclei. Examination of the replicative dependency of these enzyme activities following partial hepatectomy revealed pre-replicative elevations which were distinct for each matrix-bound enzyme. A second late-replicative peak in DNA methylase is consistent with a role of this matrix-bound enzyme in the maintenance of the inheritable methylation pattern. Mild sonication resulted in a significant release of all of these activities except RNase H. A major portion of the matrix-solubilized DNA polymerase alpha, DNA primase, 3'-5' exonuclease, and DNA methylase activities cosedimented on sucrose gradients between approximately 8-12 S. Our results are consistent with the organization of at least a portion of these replicative enzymes into nuclear matrix-bound replicational complexes. We also propose a novel pre-replicative assembly model of the matrix-bound replicational apparatus in which DNA primase plays an initial and critical role.
...
PMID:Pre-replicative association of multiple replicative enzyme activities with the nuclear matrix during rat liver regeneration. 302 82

Human SY5Y neuroblastoma cells which were differentiated in culture by treatment with 7S murine nerve growth factor for 5 weeks and selection with aphidicolin (L. Jensen, Dev. Biol. 120:56-64, 1987) demonstrated a considerably slower rate of removal of DNA adducts of benzo[a]pyrene, benzo[a]pyrenediolepoxide, and N7-methylguanine than did undifferentiated mitotic cells. A dramatic decline in unscheduled DNA synthesis induced by UV radiation was similarly observed. DNA polymerase beta and uracil DNA glycosylase were unchanged after differentiation, DNA polymerase alpha and DNA methylase decreased roughly threefold, and total apurinic-apyrimidinic endonuclease activity increased roughly threefold after treatment.
...
PMID:A reduced rate of bulky DNA adduct removal is coincident with differentiation of human neuroblastoma cells induced by nerve growth factor. 314 94

O4-Alkylthymine-DNA adducts have been implicated as causative lesions in chemical mutagenesis and carcinogenesis. To directly assess the mutagenic potential of these adducts in vivo, we have designed an enzymatic technique for introducing nucleotide analogues at predetermined sites of biologically active DNA. Escherichia coli DNA polymerase I was used in vitro to incorporate a single O4-methylthymine residue at the 3' terminus of an oligonucleotide primer opposite the adenine residue of the amber codon in bacteriophage phi X174 am3 DNA. After further extension of the primer with unmodified nucleotides, the partial-duplex product was transfected into E. coli spheroplasts. Replication of the site-specifically methylated DNA in E. coli deficient in O4-methylthymine-DNA methyltransferase (ada-) yielded 10-fold more mutant progeny phage than replication of nonmethylated DNA; no increase in mutation frequency was observed after replication in repair-proficient (ada+) E. coli. The DNA from 20 independently isolated mutant plaques all contained A.T----G.C transitions at the original site of O4-methylthymine incorporation. These data demonstrate that O4-methylthymine induces base-substitution mutations in E. coli and suggest that this adduct may be involved in mutagenesis by N-nitroso methylating agents. This enzymatic technique for site-specific mutagenesis provides an alternative to the chemical synthesis of oligonucleotides containing altered bases.
...
PMID:Mutagenic potential of O4-methylthymine in vivo determined by an enzymatic approach to site-specific mutagenesis. 346 67

In order to understand further the molecular mode of action of 5-Aza-2'-deoxycytidine (5-AZA-dCyd), a potent antileukemic agent, we prepared enzymatically 5-Aza-2'-deoxycytidine 5'-triphosphate (5-AZA-dCTP) and performed studies with purified DNA polymerase alpha and DNA methylase from mammalian cells. DNA polymerase alpha catalyzed the incorporation of 5-AZA-dCTP into DNA. The apparent Km value for 5-AZA-dCTP was estimated to be 3.0 microM; the Km of dCTP was 2.0 microM. The apparent Vmax of 5-AZA-dCTP was slightly lower than that for dCTP. 5-AZA-dCTP was a weak competitive inhibitor (Ki 4.3 microM) with respect to dCTP. Template studies with 5-AZA-dCTP showed that this nucleotide analogue was incorporated into poly(dIC), but not into poly(dAT), suggesting that the incorporation follows the rules of Watson-Crick base pairing. Incorporation of 5-AZA-dCTP into hemimethylated DNA produced a significant inhibition of DNA methylase. These results show that 5-AZA-dCTP is a very good substrate for DNA polymerase alpha and that its incorporation into DNA inhibits DNA methylation.
...
PMID:Incorporation of 5-Aza-2'-deoxycytidine-5'-triphosphate into DNA. Interactions with mammalian DNA polymerase alpha and DNA methylase. 619 Nov 92

A readily sedimentable nuclear fraction from Chinese hamster embryo fibroblast (CHEF/18) cells catalyzes incorporation of 14C-rCDP into DNA. Data indicated that this incorporation is made possible by the conversion of rCDP into a small and functionally compartmentalized, rather than a large and freely diffusible, pool of dCTP. This catalytically active sedimentable fraction from S phase CHEF/18 cells or actively replicating calf thymus cells contains nascent and template DNA, and numerous enzymes required for DNA biosynthesis including ribonucleoside diphosphate reductase, thymidylate synthetase, dihydrofolate reductase, DNA methylase, topoisomerase and DNA polymerase. We have named this catalytically active macromolecule the replitase. The replitase fraction contained spherical particles with a diameter of approximately 24 to 30 nm and had an estimated molecular weight on the order of 5 X 10(6).
...
PMID:Rapid incorporation of label from ribonucleoside disphosphates into DNA by a cell-free high molecular weight fraction from animal cell nuclei. 629 95

At an early purification stage, DNA polymerase alpha holoenzyme from calf thymus can be separated into four different forms by chromatography on DEAE-cellulose. All four enzyme forms (termed A, B, C, and D) are capable of replicating long single-stranded DNA templates, such as parvoviral DNA or primed M13 DNA. Peak A possesses, in addition to the DNA polymerase alpha, a double-stranded DNA-dependent ATPase, as well as DNA topoisomerase type II, 3'-5' exonuclease, and RNase H activity. Peaks B, C, and D all contain, together with DNA polymerase alpha, activities of primase and DNA topoisomerase type II. Furthermore, peak B is enriched in an RNase H, and peaks C and D are enriched in a 3'-5' exonuclease. DNA methylase (DNA methyltransferase) was preferentially identified in peaks C and D. Velocity sedimentation analyses of the four peaks gave evidence of unexpectedly large forms of DNA polymerase alpha (greater than 11.3 s), indicating that copurification of the above putative replication enzymes is not fortuitous. With moderate and high concentrations of salt, enzyme activities cosedimented with DNA polymerase alpha. Peak C is more resistant to inhibition by salt and spermidine than the other three enzyme forms. These results suggest the existence of a leading strand replicase (peak A) and several lagging strand replicase forms (peaks B, C, and D). Finally, the salt-resistant C form might represent a functional DNA polymerase alpha holoenzyme, possibly fitting in a higher-order structure, such as the replisome or even the chromatin.
...
PMID:Mammalian DNA polymerase alpha holoenzymes with possible functions at the leading and lagging strand of the replication fork. 658 75

Analysis of 94 kb of DNA, located between map positions 88 and 182 kb in the 330-kb chlorella virus PBCV-1 genome, revealed 195 open reading frames (ORFs) 65 codons or longer. One hundred and five of the 195 ORFs were considered major ORFs. Twenty-six of the 105 major ORFs resembled genes in the databases including three chitinases, a chitosanase, three serine/threonine protein kinases, two additional protein kinases, a tyrosine protein phosphatase, two ankyrins, an ornithine decarboxylase, a copper/zinc-superoxide dismutase, a proliferating cell nuclear antigen, a DNA polymerase, a fibronectin-binding protein, the yeast Ski2 protein, an adenine DNA methyltransferase and its corresponding DNA site-specific endonuclease, and an amidase. The genes for the 105 major ORFs were evenly distributed along the genome and, except for one noncoding 1788-nucleotide stretch, the genes were close together. Unexpectedly, a 900-bp region in the 1788-bp noncoding sequence resembled a CpG island.
...
PMID:Analysis of 94 kb of the chlorella virus PBCV-1 330-kb genome: map positions 88 to 182. 861 77

Lymphocystis disease virus (LCDV) is the causative agent of lymphocystis disease, which has been reported to occur in over 100 different fish species worldwide. LCDV is a member of the family Iridoviridae and the type species of the genus Lymphocystivirus. The virions contain a single linear double-stranded DNA molecule, which is circularly permuted, terminally redundant, and heavily methylated at cytosines in CpG sequences. The complete nucleotide sequence of LCDV-1 (flounder isolate) was determined by automated cycle sequencing and primer walking. The genome of LCDV-1 is 102.653 bp in length and contains 195 open reading frames with coding capacities ranging from 40 to 1199 amino acids. Computer-assisted analyses of the deduced amino acid sequences led to the identification of several putative gene products with significant homologies to entries in protein data banks, such as the two major subunits of the viral DNA-dependent RNA polymerase, DNA polymerase, several protein kinases, two subunits of the ribonucleoside diphosphate reductase, DNA methyltransferase, the viral major capsid protein, insulin-like growth factor, and tumor necrosis factor receptor homolog.
...
PMID:The complete DNA sequence of lymphocystis disease virus. 914 76

Transformed cells are characterized by imbalances in metabolic routes. In particular, different key enzymes of nucleotide metabolism and DNA biosynthesis, such as CTP synthetase, thymidylate synthase, dihydrofolate reductase, IMP dehydrogenase, ribonucleotide reductase, DNA polymerase, and DNA methyltransferase, are markedly up-regulated in certain tumor cells. Together with the concomitant down-modulation of the purine and pyrimidine degradation enzymes, the increased anabolic propensity supports the excessive proliferation of transformed cells. However, many types of cancer cells have maintained the ability to differentiate terminally into mature, non-proliferating cells not only in response to physiological receptor ligands, such as retinoic acid, vitamin D metabolites, and cytokines, but also following exposure to a wide variety of non-physiological agents such as antimetabolites. Interestingly, induction of tumor cell differentiation is often associated with reversal of the transformation-related enzyme deregulations. An important class of differentiating compounds comprises the antimetabolites of purine and pyrimidine nucleotide metabolism and nucleic acid synthesis, the majority being structural analogs of natural nucleosides. The CTP synthetase inhibitors cyclopentenylcytosine and 3-deazauridine, the thymidylate synthase inhibitor 5-fluoro-2'-deoxyuridine, the dihydrofolate reductase inhibitor methotrexate, the IMP dehydrogenase inhibitors tiazofurin, ribavirin, 5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR) and mycophenolic acid, the ribonucleotide reductase inhibitors hydroxyurea and deferoxamine, and the DNA polymerase inhibitors ara-C, 9-(2-phosphonylmethoxyethyl)adenine (PMEA), and aphidicolin, as well as several nucleoside analogs perturbing the DNA methylation pattern, have been found to induce tumor cell differentiation through impairment of DNA synthesis and/or function. Thus, by selectively targeting those anabolic enzymes that contribute to the neoplastic behavior of cancer cells, the normal cellular differentiation program may be reactivated and the malignant phenotype suppressed.
...
PMID:Role of antimetabolites of purine and pyrimidine nucleotide metabolism in tumor cell differentiation. 1041 91


1 2 Next >>

---