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)

The tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is present in tobacco smoke and is hepatocarcinogenic in rats. Its bioactivation in rat hepatocytes leads to methylation and pyridyloxobutylation of DNA. Rat hepatocytes were cultured in serum-free William medium E on collagen-coated dishes. We demonstrated that some enzymes of the base and/or excision-repair pathways were involved in repair of NNK-induced DNA damage, measured by [methyl-3H] thymidine incorporation. Unscheduled DNA synthesis (UDS) induced by N-methyl-N-nitrosourea (MNU), NNK, N'-nitrosonornicotine (NNN) and 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc) increased 2.9-, 2.8-, 1.5- and 3.5-fold, respectively, suggesting that methylated and/or pyridyloxobutylated-DNA by these four nitroso compounds is repaired by the excision pathway. Moreover, levels of NNK-induced UDS were dose (1-3 mM) and time (1-18 h) dependent. Enzymes involved in the excision repair pathways were selectively inhibited. Inhibitors of DNA topoisomerase I (camptothecin) and topoisomerase II (etoposide, nalidixic acid) did not decrease the induction of UDS, suggesting that topoisomerases are not involved in the repair of NNK-induced damage. While aphidicolin and arabinocytidine (DNA polymerase alpha, delta, epsilon inhibitors) totally inhibited NNK- and NNKOAc-induced UDS, dideoxythymidine (DNA polymerase beta inhibitor) inhibited NNK- and NNKOAc-induced UDS by 40 and 33%, respectively. We conclude that DNA polymerase alpha, delta or epsilon and to a lesser degree polymerase beta are involved in the repair of pyridyloxobutylated DNA. Previous studies showed that inhibition of poly(ADP-ribosyl) polymerase (PARP) by 3-aminobenzamide (3-ab) facilitated DNA ligation. Our results demonstrate that 3-ab increased NNK-induced UDS, but does not affect NNKOAc-induced UDS. These observations suggest that the ligation step is rate limiting in the repair of methylated DNA but not of pyridyloxobutylated DNA.
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PMID:Modulation of DNA repair by various inhibitors of DNA synthesis following 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induced DNA damage. 956 22

Poly(ADP-ribose) polymerase (PARP) is a component of the multiprotein DNA replication complex (MRC, DNA synthesome) that catalyzes replication of viral DNA in vitro. PARP poly(ADP-ribosyl)ates 15 of the approximately 40 proteins of the MRC, including DNA polymerase alpha (DNA pol alpha), DNA topoisomerase I (topo I), and proliferating-cell nuclear antigen (PCNA). Although about equal amounts of MRC-complexed and free forms of PCNA were detected by immunoblot analysis of HeLa cell extracts, only the complexed form was poly(ADP-ribosyl)ated, suggesting that poly(ADP-ribosyl)ation of PCNA may regulate its function within the MRC. NAD inhibited the activity of DNA pol delta in the MRC in a dose-dependent manner, whereas the PARP inhibitor, 3-AB, reversed this inhibitory effect. The roles of PARP in modulating the composition and enzyme activities of the DNA synthesome were further investigated by characterizing the complex purified from 3T3-L1 cells before and 24 h after induction of a round of DNA replication required for differentiation of these cells; at the latter time point, approximately 95% of the cells are in S phase and exhibit a transient peak of PARP expression. The MRC was also purified from similarly treated 3T3-L1 cells depleted of PARP by antisense RNA expression; these cells do not undergo DNA replication nor terminal differentiation. Both PARP protein and activity and essentially all of the DNA pol alpha and delta activities exclusively cosedimented with the MRC fractions from S phase control cells, and were not detected in the MRC fractions from PARP-antisense or uninduced control cells. Immunoblot analysis further revealed that, although PCNA and topo I were present in total extracts from both control and PARP-antisense cells, they were present in the MRC fraction only from induced control cells, indicating that PARP may play a role in their assembly into an active DNA synthesome. In contrast, expression of DNA pol alpha, DNA primase, and RPA was down-regulated in PARP-antisense cells, suggesting that PARP may be involved in the expression of these proteins. Depletion of PARP also prevented induction of the expression of the transcription factor E2F-1, which positively regulates transcription of the DNA pol alpha and PCNA genes; thus, PARP may be necessary for expression of these genes when quiescent cells are stimulated to proliferate.
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PMID:Regulation of the expression or recruitment of components of the DNA synthesome by poly(ADP-ribose) polymerase. 964 17

Previously, we have purified and characterized DNA helicase III from the yeast Saccharomyces cerevisiae [Shimizu, K. and Sugino, A. (1993) J. Biol. Chem. 268, 9578-9584]. Here, we have further characterized DNA helicase III activity. It was found that the combined action of the helicase III, yeast DNA topoisomerase I (yTop I), and yeast RPA protein on a covalently closed, circular DNA generates a highly underwound DNA species that has been called form I* or form U. Furthermore, these underwound structures can be accessed by yeast DNA polymerase I (alpha)-primase to initiate DNA synthesis. These reactions mimic in vivo initiation of chromosomal DNA replication. In order to clone the gene encoding DNA helicase III, a partial amino acid sequence of the purified DNA helicase III polypeptide was determined. Using a mix oligonucleotides synthesized based on the amino acid sequence of the helicase, we cloned the gene encoding the helicase III and found it to be identical to YER176W (HEL1) on chromosome V. The amino acid sequence predicted from the nucleotide sequence of the gene has conserved DNA helicase domains that are highly homologous to those of DNA helicases required for DNA replication. However, complete deletion of the gene from the chromosome did not result in any growth defect, suggesting that the gene product is not required for DNA synthesis or that it is functionally substituted by other helicase(s). Furthermore, the deletion strain does not exhibit sensitivity to any DNA-damaging reagents, although it is hypersensitive to calcofluor white, hygromycin, and papulacandin.
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PMID:DNA helicase III of Saccharomyces cerevisiae, encoded by YER176w (HEL1), highly unwinds covalently closed, circular DNA in the presence of a DNA topoisomerase and yRF-A. 999 Jan 19

Cytoplasmic extracts from early-passage (young), late-passage (senescent) normal human fibroblast (HF) cultures and immortalized human cell lines (HeLa, HT-1080, and MANCA) were analyzed for their ability to support semiconservative DNA synthesis in an in vitro SV40-ori DNA replication system. Unsupplemented extracts from the three permanent cell lines were demonstrated to be active in this system; whereas young HF extracts were observed to be minimally active, and no activity could be detected in the senescent HF extracts. The activity of these extracts was compared after supplementation with three recombinant human replication factors: (1) the catalytic subunit of DNA polymerase alpha (DNA pol-alpha-cat), (2) the three subunits of replication protein A (RPA), and (3) DNA topoisomerase I (Topo I). The addition of all three recombinant proteins is required for optimum activity in the young and senescent HF extracts; the order of the level of activity is: transformed > young HF > senescent HF. Young HF extracts supplemented with RPA alone are able to support significant replicative activity but not senescent extracts which require both RPA and DNA pol-alpha-cat for any detectable activity. The necessary requirement for these factors is confirmed by the failure of unsupplemented young and senescent extracts to activate MANCA extracts that have been immunodepleted of DNA pol-alpha-cat or RPA. Immunocytochemical studies revealed that RPA, DNA pol-alpha, PCNA, and topo I levels are higher in the immortal cell types used in these studies. In the HF cells, levels of DNA pol-alpha-cat and PCNA are higher (per mg protein) in the low-passage than in the senescent cells. By contrast, RPA levels, as determined by immunocytochemical or Western blot studies, were observed to be similar in both young and senescent cell nuclei. Taken together, these results indicate that the low to undetectable activity of young HF extracts in this system is due mainly to reduced intracellular levels of RPA, while the senescent HF extracts are relatively deficient in DNA polymerase alpha and probably some other essential replication factors, as well as RPA. Moreover, the retention of RPA in the senescent HF nuclei contributes to the low level of this factor in the cytoplasmic extracts from these cells.
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PMID:Analysis of the capacity of extracts from normal human young and senescent fibroblasts to support DNA synthesis in vitro. 1022 81

Camptothecins demonstrate a broad spectrum of antitumor activity. Although they are known to trap DNA topoisomerase I on DNA, form cleavable complexes, and generate DNA breaks upon collision with DNA or RNA polymerases, the precise mechanisms predictive for antitumor activity remain to be identified. Recent studies using panels of colorectal and breast cancer cell lines indicate that events downstream of cleavable complexes are more relevant. In this study, we chose SN-38, an active metabolite of irinotecan, to characterize DNA double strand breaks and repair mechanisms induced by this type of drugs using a human head and neck squamous cell carcinoma cell line A253. The results showed that 2-h exposure of cells to an IC(50) concentration of SN-38 induces biphasic DNA double-strand break (DSBs): an immediate phase, which was greatly reduced within 8 h, and a lagging phase, culminating 24 h after drug removal. Three DNA double-strand break repair protein complexes were activated: DNA-dependent protein kinase (DNA-PK), NBS1-MRE11-RAD50, and BRCA1. Aphidicolin, a DNA polymerase inhibitor, abolished both phase I DSBs and the activation of repair protein complexes, suggesting that they resulted from the collision between the cleavable complex and DNA polymerase of S-phase cells. This is in contrast to ionizing radiation-induced activation of DNA-PK and NBS1-MRE11-RAD50 complexes that occur predominantly among non-S-phase cells. The trigger for phase II DSBs cannot be abolished by aphidicolin. The data also indicate that DNA fragments in the size of 50 to 200 kilobases were detected in the lagging phase. This suggests that the late DNA DSBs were associated with apoptotic cell death.
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PMID:Induction of biphasic DNA double strand breaks and activation of multiple repair protein complexes by DNA topoisomerase I drug 7-ethyl-10-hydroxy-camptothecin. 1190 Dec 12

To clarify the mechanisms of fish fertilization, the effects of inhibitors of DNA polymerase-alpha and DNA topoisomerases on nuclear behavior before and after fertilization were examined in eggs of the medaka, Oryzias latipes. Eggs underwent the fertilization process from sperm penetration to karyogamy of pronuclei, even when inseminated and incubated in the continuous presence of aphidicolin (DNA polymerase alpha inhibitor), camptothecin (DNA topoisomerase I inhibitor), etoposide, or beta-lapachone (DNA topoisomerase II inhibitor). However, continuous treatment with aphidicolin or camptothecin during fertilization inhibited the formation of sister chromosomes that were normally separated into blastomeres at the time of the subsequent cleavage. Sister chromosome formation appeared concomitantly with an increase in histone H1 kinase activity at the end of DNA synthesis, 30 min post insemination. However, non-activated eggs that were inseminated in saline containing anesthetic MS222 and aphidicolin had high levels of histone H1 kinase and MAP kinase activities, and transformation of the penetrated sperm nucleus to metaphase chromosomes occurred even in the presence of aphidicolin or camptothecin. The male chromosomes were normally separated into two anaphase chromosome masses upon egg activation. These results suggest that DNA polymerase alpha or DNA topoisomerase I, but not DNA topoisomerase II, may be required for the process by which the mitotic interphase nucleus transforms to separable metaphase chromosomes while the activity of MAP kinase is low, unlike the situation in meiotic division, during which MAP kinase activity is high and DNA replication is not required.
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PMID:Studies on fertilization in the teleost IV. Effects of aphidicolin and camptothecin on chromosome formation in fertilized medaka eggs. 1217 64

Two flavonoid glycosides, kaempferol 3-O-(6"-acetyl)-beta-glucopyranoside (KAG) and quercetin 3-O-(6"-acetyl)-beta-glucopyranoside (QAG), were found to be inhibitors of eukaryotic DNA polymerases from a Japanese vegetable, Petasites japonicus. These compounds inhibited the activities of mammalian replicative DNA polymerases (i.e., pol alpha, delta, and epsilon), but not other pol beta, eta, kappa, and lambda activities. KAG was a stronger inhibitor and more selective to pol alpha than QAG. The IC(50) values of KAG for pol alpha, delta, and epsilon were 41, 164, and 127 microM, respectively. The pol alpha inhibition by KAG was non-competitive with respect to both the DNA template-primer and the dNTP substrate. KAG and QAG did not influence the activities of prokaryotic DNA polymerases or other mammalian DNA metabolic enzymes such as human immunodeficiency virus type 1 reverse transcriptase, human telomerase, human DNA topoisomerase I and II, T7 RNA polymerase, and bovine deoxyribonuclease I. Therefore, we concluded that these flavonoid glycosides are moderate replicative DNA polymerase inhibitors leaning more relatively to pol alpha, and could be used as chromatographic carriers to purify the DNA polymerases rather than cytotoxic agents. We then made a KAG-conjugated column such as the epoxy-activated Sepharose 6B. In the column, pol alpha was selectively adsorbed and eluted.
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PMID:Flavonoid glycoside: a new inhibitor of eukaryotic DNA polymerase alpha and a new carrier for inhibitor-affinity chromatography. 1256 87

We found a novel inhibitor specific to eukaryotic DNA polymerase epsilon(pol epsilon) from plant cultured cells, Nicotina tabacum L. The compound (compound 1) was a dipeptide alcohol, L-homoserylaminoethanol. The 50% inhibition of pol epsilon activity by the compound was 43.6 microg/mL, and it had almost no effect on the activities of the other eukaryotic DNA polymerases such as alpha, beta, gamma and delta, prokaryotic DNA polymerases, nor DNA metabolic enzymes such as human telomerase, human immunodeficiency virus type 1 reverse transcriptase, T7 RNA polymerase, human DNA topoisomerase I and II, T4 polynucleotide kinase and bovine deoxyribonuclease I. Kinetic studies showed that inhibition of pol epsilon by the compound was non-competitive with respect to both template-primer DNA and nucleotide substrate. We succeeded in chemically synthesizing the stereoisomers, L-homoserylaminoethanol and D-homoserylaminoethanol, and found both were effective to the same extent. The IC(50) values of L- and D-homoserylaminoethanols for pol epsilon were 42.0 and 41.5 microg/mL, respectively. This represents the second discovery of a pol epsilon-specific inhibitor, and the first report on a water-soluble peptide-like compound as the inhibitor, which is required in biochemical studies of pol epsilon.
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PMID:L-Homoserylaminoethanol, a novel dipeptide alcohol inhibitor of eukaryotic DNA polymerase from a plant cultured cells, Nicotina tabacum L. 1498 Jun 8

We studied the cytotoxic effects of various DNA replication inhibitors on MMR-deficient and -proficient colon carcinoma cell lines. DNA polymerase (pol) inhibitors including aphidicolin and gemcitabine, and hydroxyurea were more toxic (1.7 to 2.8-fold) to hMLH1-deficient HCT116 than to hMLH1-proficient HCT116+ch3. Similarly, pol inhibitors were more toxic to hMSH2-deficient LoVo than to hMSH2-proficient LoVo+ch2. In contrast, DNA topoisomerase I inhibitors, such as CPT-11, SN-38, and topotecan, were more toxic to MMR-proficient cells. Our results suggest that MMR-deficient colon carcinoma cells are hypersensitive to inhibitors of the pol reaction.
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PMID:Hypersensitivity in DNA mismatch repair-deficient colon carcinoma cells to DNA polymerase reaction inhibitors. 1573 91

Novel poxviruses were identified in skin lesions of several species of cetaceans and pinnipeds using polymerase chain reaction targeting DNA polymerase and DNA topoisomerase I genes of members of the subfamily Chordopoxvirinae. With the exception of parapoxviruses, no molecular data of marine mammal poxviruses were available to infer genetic and evolutionary relatedness to terrestrial vertebrate poxviruses. Viruses were assigned to a cetacean poxvirus 1 (CPV-1) group based on nucleotide and amino acid identities of gene fragments amplified from skin lesions of Asian bottlenose (Tursiops aduncus), Atlantic bottlenose (Tursiops truncatus), rough-toothed (Steno bredanensis), and striped (Stenella coeruleoalba) dolphins. A different poxvirus was detected in skin lesions of a bowhead whale (Balaena mysticetus) and provisionally assigned to a CPV-2 group. These viruses showed highest identity to terrestrial poxviruses of the genera Orthopoxvirus and Suipoxvirus. A novel species-specific poxvirus was also identified in skin lesions of Steller sea lions (Eumetopias jubatus). None of these poxviruses were found to have amplifiable hemagglutinin gene sequences. Novel parapoxviruses were also identified in skin lesions of Steller sea lions and spotted seals (Phoca largha). A significant degree of divergence was observed in sequences of Steller sea lion parapoxviruses, while those of spotted seals and harbor seals (Phoca vitulina) were highly conserved.
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PMID:Genetic identification of novel poxviruses of cetaceans and pinnipeds. 1632 32


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