Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An assay has been developed to measure the ability of human lymphocytes to repair damage to DNA. In this assay, purified human lymphocytes are exposed to graded doses of radiation and then stimulated with phytohemagglutinin to undergo DNA replication. The rate of incorporation of thymidine in irradiated lymphocytes during the second and subsequent rounds of DNA replication is taken to be indicative of the ability of the cells to repair damage to DNA. In lymphocytes from normal individuals, X-irradiation with doses of 100 to 800 rads was found to inhibit phytohemagglutinin-stimulated thymidine incorporation proportionally to the dose of radiation without curtailing the induction of DNA polymerase. The response to phytohemagglutinin of lymphocytes from a patient with xeroderma pigmentosum after exposure to graded doses of X-irradiation was found to be similar to that of the normal controls, whereas the response after ultraviolet irradiation was markedly impaired. In contrast, lymphocytes from patients with ataxia telangiectasia were hypersensitive to X-irradiation. The data on these clinical syndromes support the idea that this assay measures DNA repair and indicates the feasibility of using this method for screening individuals for genetic deficits in DNA repair.
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PMID:Screening for deficits in DNA repair by the response of irradiated human lymphocytes to phytohemagglutinin. 90 9

Ataxia telangiectasia, Bloom's syndrome and normal fibroblasts were compared as to the capacity of their cellular extracts to enhance the priming activity of gamma-irradiated colicin E1 DNA for purified DNA polymerase. It was found that an ataxia strain had substantially lower, and a Bloom's syndrome strain had slightly lower capacity than a normal strain; while the activities of apurinic site specific endonuclease in these extracts were comparable.
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PMID:DNA repair enzymes in ataxia telangiectasia and Bloom's syndrome fibroblasts. 92 14

DNA polymerase beta levels were measured in 4 cell lines of normal human skin fibroblasts and in 5 cell lines of skin fibroblasts from patients with ataxia telangiectasia, an autosomal recessive disease exhibiting marked X-ray sensitivity. The enzyme specific activities for the normal lines were similar and the mean value was 2-fold lower than the mean value for the ataxia lines. With both kinds of cells, the enzyme level did not change as the cultures progressed from logarithmic to stationary phase of growth. Thus, this putative DNA repair enzyme appears to be 'constitutive' in human skin fibroblast lines, and a modest elevation of beta-polymerase activity is associated with ataxia telangiectasia. These results are discussed in the context to current views about DNA-repair enzymes in X-ray-sensitive cultured mammalian cells.
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PMID:Measurement of DNA polymerase beta in skin fibroblast cell lines from patients with ataxia telangiectasia. 405 46

Three ataxia telangiectasia homozygotes, one heterozygote and normal fibroblast strains were compared as to the capacity of their cellular extracts to enhance the priming activity of gamma-irradiated colicin E1 DNA for purified DNA polymerase (EC 2.7.7.7) of Escherichia coli. It was found that homozygotes had substantially lower activity than normal strains, while no difference was detected between the heterozygote and normal strains. In vitro complementation of the activity occurred between extracts of certain strains of homozygotes, allocating them to two complementation groups.
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PMID:DNA repair enzyme deficiency and in vitro complementation of the enzyme activity in cell-free extracts from ataxia telangiectasia fibroblasts. 626 84

The antibiotic, aphidicolin, is a potent inhibitor of DNA polymerase alpha and consequently of de novo DNA synthesis in human cells. We report here that in gamma-irradiated normal human cells, aphidicolin (at 5 micrograms/ml and less) had no significant effect on the rate of the rejoining of DNA single strand breaks or rate of removal of DNA lesions assayed as sites sensitive to incising activities present in crude protein extracts of Micrococcus luteus cells. gamma-irradiated human ataxia telangiectasia cells are known to demonstrate enhanced cell killing and exhibit resistance to the inhibiting effects of radiation on DNA synthesis. Under conditions of minimal aphidicolin cytotoxicity but extensive inhibition of de novo DNA synthesis, the radiation responses of neither normal nor ataxia telangiectasia cells were significantly modified by aphidicolin. Firstly, we conclude that human DNA polymerase alpha is not primarily involved in the repair of the two classes of radiogenic DNA lesions examined. Secondly, the radiation hypersensitivity of ataxia telangiectasia cells cannot be explained on the basis of premature replication of damaged cellular DNA resulting from the resistance of de novo DNA synthesis to inhibition by ionizing radiation.
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PMID:Effect of aphidicolin on de novo DNA synthesis, DNA repair and cytotoxicity in gamma-irradiated human fibroblasts. Implications for the enhanced radiosensitivity in ataxia telangiectasia. 640 35

An enzyme that enhances the activity of DNA polymerase I (EC 2.7.7.7) for gamma-irradiated calf thymus DNA was demonstrated in cellular extracts of normal human fibroblasts and lymphoid-cell lines. This enzyme was found to be deficient in all cellular extracts of fibroblasts and lymphoid-cell lines examined from patients with the autosomal recessive disease ataxia telangiectasia. The activity in cellular extracts from normal fibroblasts was removed when heated to 100 degrees C for 2 min or when the assay was performed at 4 degrees C. No significant deficiency in primer-activating enzyme activity was observed in cell-free extracts of lymphoid lines from patients with xeroderma pigmentosum, Huntington's chorea or neurofibromatosis, or from an ataxia telangiectasia heterozygote.
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PMID:An enzyme activity in normal and ataxia telangiectasia cell lines which is involved in the repair of gamma-irradiation-induced DNA damage. 645 Dec 16

The regulation of DNA repair during serum stimulation of quiescent cells was examined in normal human cells, in fibroblasts from three xeroderma pigmentosum complementation groups (A, C, and D), in xeroderma pigmentosum variant cells, and in ataxia telangiectasia cells. The regulation of nucleotide excision repair was examined by exposing cells to ultraviolet irradiation at discrete intervals after cell stimulation. Similarly, base excision repair was quantitated after exposure to methylmethane sulfonate. WI-38 normal human diploid fibroblasts, xeroderma pigmentosum variant cells, as well as ataxia telangiectasia cells enhanced their capacity for both nucleotide excision repair and for base excision repair prior to their enhancement of DNA synthesis. Further, in each cell strain, the base excision repair enzyme uracil DNA glycosylase was increased prior to the induction of DNA polymerase using the identical cells to quantitate each activity. In contrast, each of the three xeroderma complementation groups that were examined failed to increase their capacity for nucleotide excision repair above basal levels at any interval examined. This result was observed using either unscheduled DNA synthesis in the presence of 10 mM hydroxyurea or using repair replication in the absence of hydroxyurea to quantitate DNA repair. However, each of the three complementation groups normally regulated the enhancement of base excision repair after methylmethane sulfonate exposure and each induced the uracil DNA glycosylase prior to DNA synthesis. These results suggest that there may be a relationship between the sensitivity of xeroderma pigmentosum cells from each complementation group to specific DNA damaging agents and their inability to regulate nucleotide excision repair during cell stimulation.
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PMID:Regulation of DNA repair in serum-stimulated xeroderma pigmentosum cells. 648 Jun 91

The relationship between the repair processes occuring at the G2 phase of the cell cycle and cytogenetic damage in ataxia telangiectasia (AT) cells was studied. Lymphoblastoid cells derived from normal, heterozygote AT (HzAT) and three AT patients were exposed to X-rays or fission neutrons and post-treated with inhibitors of DNA synthesis/repair, such as inhibitors of DNA polymerases alpha, delta and epsilon (cytosine arabinoside, ara-C; aphidicolin, APC; buthylphenylen-guanine, BuPdG) or ribonucleotide reductase (hydroxyurea, HU). A strong increase of radiation-induced chromosomal aberrations was observed in normal and HzAT cells post-treated with ara-C, APC and HU, but not in the presence of BuPdG. No enhancing effect was observed in cells derived from AT patients, except for HU post-irradiation treatment. These results suggest that the enzymes that can be inhibited by these agents are not directly involved in the repair of radiation damage induced in G2 cells from AT patients, indicating that probably the AT cells that we used lack the capability to transform the primary DNA lesions into reparable products, or that AT cells might contain a mutated form of DNA polymerase resistant to the inhibitors.
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PMID:Lack of effect of inhibitors of DNA synthesis/repair on the ionizing radiation-induced chromosomal damage in G2 stage of ataxia telangiectasia cells. 793 Aug 33

Mutation of the essential Schizosaccharomyces pombe rad4/cut5 gene causes sensitivity to UV and ionising radiation at the permissive temperature whilst at the restrictive temperature cells fail to undergo DNA replication but still attempt mitosis owing to a defective S-phase checkpoint response. Many mutations in genes encoding DNA replication proteins also abolish checkpoint responses, possibly because the replication machinery is a pre-requisite for the generation of the signal. We demonstrate here that rad4/cut5 cells fail to arrest cell division when treated with the replication inhibitor hydroxyurea at the semi-permissive temperature 32 degrees C, but retain essentially normal replicative capacity. This demonstrates that the replication and checkpoint function of the rad4/cut5 gene product can be separated and that the Rad4 protein differs from other replication proteins in being directly involved in generating the S-phase checkpoint signal. Furthermore, we have investigated the checkpoint response or rad4/cut5-deficient cells to gamma-irradiation and UV-mimetic drugs. We find that, at the restrictive temperature, the rad4-/cut5- cells fail to delay mitosis in response to gamma-irradiation whilst retaining a normal checkpoint response to the UV-mimetic drug 4-nitroquinoline-1-oxide. The lack of the gamma-irradiation checkpoint is reminiscent of the deficiency associated with mutation of the human ATM locus, the causative deficiency of the heritable disorder ataxia telangiectasia. The implications of our results for the organisation of distinct checkpoint-response pathways in both fission yeast and mammalian cells are discussed. Moreover the data are consistent with a model in which the generation of the S-Phase checkpoint signal is DNA polymerase epsilon dependent.
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PMID:Characterisation of the Schizosaccharomyces pombe rad4/cut5 mutant phenotypes: dissection of DNA replication and G2 checkpoint control function. 926 24

DNA replication origins are located at random with respect to DNA sequence in Xenopus early embryos and on DNA replicated in Xenopus egg extracts. We have recently shown that origins fire throughout the S phase in Xenopus egg extracts. To study the temporal regulation of origin firing, we have analyzed origin activation in sperm nuclei treated with the DNA polymerase inhibitor aphidicolin. Sperm chromatin was incubated in Xenopus egg extracts in the presence of aphidicolin and transferred to a fresh extract, and digoxigenin-dUTP and biotin-dUTP were added at various times after aphidicolin release to selectively label early and late replicating DNA. Molecular combing analysis of single DNA fibers showed that only a fraction of potential origins were able to initiate in the presence of aphidicolin. After release from aphidicolin, the remaining origins fired asynchronously throughout the S phase. Therefore, initiation during the S phase depends on the normal progression of replication forks assembled at earlier activated origins. Caffeine, an inhibitor of the checkpoint kinases ATR and ATM, did not relieve the aphidicolin-induced block to origin firing. We conclude that a caffeine-insensitive intra-S phase checkpoint regulates origin activation when DNA synthesis is inhibited in Xenopus egg extracts.
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PMID:Aphidicolin triggers a block to replication origin firing in Xenopus egg extracts. 1127 43


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