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)

DNA polymerase delta, a fourth cellular DNA polymerase, might play an important role in cellular events. The properties of the enzyme suggested possible roles in nuclear DNA replication and DNA repair. By using specific assays that allow the determination of DNA polymerases delta and alpha in crude enzyme fractions (1), we show here that in developing rat neurons, which stop dividing before birth, both DNA polymerases delta and alpha drop sharply in an identical pattern from a high level with the approach of term and disappear at approximately three weeks of postnatal age. The results suggest that DNA polymerases delta and alpha might have a coordinated function during DNA replication in eukaryotic cells.
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PMID:Developmental activity profile of DNA polymerases delta and alpha in rat neurons suggests a coordinated in vivo function. 297 51

Metabolic activation of cyclophosphamide (CP) by microsomal mixed function hydroxylases yields 4-hydroxycyclophosphamide and aldophosphamide defined as activated CP. Activated CP shows relatively high cancerotoxic selectivity in vivo and cytotoxic specificity in vitro and can be trapped rapidly by reversible reaction of hemiaminal group of the oxazaphosphorine ring with protein thiols to form protein bound activated CP (protein-S-CP). Protein-S-CP stores activated CP in a highly stable form. From pharmacokinetics of activated CP in mice after the injection of cyclophosphamide, it was calculated that about 17% of the CP dose given was stored in a pool of protein bound activated CP lasting for several days. From therapy studies with 4-(S-ethanol)-sulfido-CP in combination with excess of cysteine, it was concluded that the protein-S-CP pool may be that form of activated CP which is mainly responsible for the specific cytotoxic effects in the tumor cells. On the other hand free unbound 4-OH-CP was shown to contribute mainly to overall toxicity. No spontaneous toxicogenation of activated CP was noted under in vivo conditions. 3'-5' Exonucleases were found to hydrolyze 4-OH-CP, yielding phosphoramide mustard and acrolein as split products. Because of the low affinity of 4-OH-CP for plain 3'-5' exonucleases, it seems however unlikely that these enzymes play a major role in the antitumor effect of CP in vivo. 3'-5' Exonucleases associated to DNA polymerase like in DNA polymerase delta from rabbit bone marrow or in DNA polymerase I from E. coli are more likely candidates for 4-OH-CP toxicogenation because of the much higher specific activity with 4-OH-CP as substrate. In experiments with DNA polymerase I from E. coli, 4-OH-CP was shown to inhibit DNA polymerase activity after toxicogenation by the 3'-5' exonuclease subsite of the enzyme. This suggests an enzyme mechanism based suicide inactivation of the DNA polymerase. Because of the close spatial cooperation of the DNA polymerase and 3'-5' exonuclease subsites with primer/template a site-specific alkylation of DNA is also postulated. Thus we raised the hypothesis that cytotoxic specificity of activated CP is based on the interaction of protein-S-CP (protein bound activated CP) with DNA polymerase/3'-5' exonuclease as the target. In a P 815 mouse mast-cell tumor we determined by means of 5' AMP agarose affinity chromatography two/third of total DNA polymerase to be associated with 3'-5' exonuclease.
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PMID:The enzymatic basis of cyclophosphamide specificity. 302 54

Purified calf thymus DNA polymerases delta I and II each have an associated 3' to 5' exonuclease but otherwise resemble DNA polymerase alpha in size, biochemical kinetic parameters, and the presence of DNA primase [Crute, J. J., Wahl, A. F., & Bambara, R. A. (1986) Biochemistry 25, 26-36]. Here we demonstrate a functional association of polymerase and exonuclease with each delta form. Furthermore, we show that the exonuclease can be dissociated from DNA polymerase delta I but does not appear to be removable from DNA polymerase delta II. Polymerases delta I, delta II, and alpha are equally sensitive to the inhibitor aphidicolin, suggesting a similarity in active site structure. In comparison with DNA polymerase alpha and delta II, DNA polymerase delta I has intermediate sensitivity to 2-(p-n-butylanilino)-2'-deoxyadenosine 5'-triphosphate (BuAdATP) or N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP). The activity of the DNA primase of the delta II enzyme is insensitive to BuAdATP whereas 1.0 microM of this inhibitor will decrease the activity of the DNA primase of the alpha and delta I enzymes approximately 50%. Two monoclonal antibodies that potently inhibit DNA polymerase alpha are only slightly inhibitory to DNA polymerase delta I and are ineffective at inhibiting DNA polymerase delta II. DNA polymerase delta II had been previously found to be nearly inactive on nuclease-treated calf thymus DNA, relative to its activity on homopolymeric DNA. We find that addition of purified calf histone proteins or spermidine can greatly enhance synthesis by this enzyme on activated calf DNA.
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PMID:Properties of two forms of DNA polymerase delta from calf thymus. 309 36

DNA polymerases delta and alpha were purified from CV-1 cells, and their sensitivities to the inhibitors aphidicolin, (p-n-butylphenyl)deoxyguanosine triphosphate (BuPdGTP), and monoclonal antibodies directed against DNA polymerase alpha were determined. The effects of these inhibitors on DNA replication in permeabilized CV-1 cells were studied to investigate the potential roles of polymerases delta and alpha in DNA replication. Aphidicolin was shown to be a more potent inhibitor of DNA replication than of DNA polymerase alpha or delta activity. Inhibition of DNA replication by various concentrations of BuPdGTP was intermediate between inhibition of purified polymerase alpha or delta activity. Concentrations of BuPdGTP which totally abolished DNA polymerase alpha activity were much less effective in reducing DNA replication, as well as the activity of DNA polymerase delta. Monoclonal antibodies which specifically inhibited polymerase alpha activity reduced, but did not abolish, DNA replication in permeable cells. BuPdGTP, as well as anti-polymerase alpha antibodies, inhibited DNA replication in a nonlinear manner as a function of time. Depending upon the initial or final rates of inhibition of replication by BuPdGTP and anti-alpha antibodies, as little as 50%, or as much as 80%, of the replication activity can be attributed to polymerase alpha. The remaining replication activity (20-50%) is tentatively attributed to polymerase delta, because it was aphidicolin sensitive and resistant to both anti-polymerase alpha antibodies and low concentrations of BuPdGTP. A concentration of BuPdGTP which abolished polymerase alpha activity reduced, but did not abolish, both the synthesis and maturation of nascent DNA fragments.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Identification of DNA polymerase delta in CV-1 cells: studies implicating both DNA polymerase delta and DNA polymerase alpha in DNA replication. 312 21

The DNA polymerase-primase from Drosophila melanogaster contains a cryptic 3'----5' exonuclease that can be detected after separation of the 182-kDa polymerase subunit from the four-subunit enzyme. To determine the specificity of excision of mispaired nucleotides by the exonuclease, we have utilized primed phi X174am3 single-stranded DNA containing a noncomplementary nucleotide at the 3'-primer terminus, opposite deoxyadenosine at position 587 in the amber3 codon of the template strand. In the absence of polymerization, the preference for excision of the mispaired nucleotide from the primer is C greater than A much greater than G. Excision under these conditions is inhibited by the addition of deoxyguanosine monophosphate. Under conditions of concomitant DNA synthesis, the preference for excision at this site becomes A = G much greater than C, and excision is insensitive to deoxyguanosine monophosphate. The high fidelity of DNA synthesis exhibited by the isolated 182-kDa polymerase subunit is not reduced by concentrations of deoxyguanosine monophosphate or adenosine monophosphate that inhibit proofreading by prokaryotic DNA polymerases. Thus, the 3'----5' exonuclease of the Drosophila DNA polymerase-primase participates in exonucleolytic proofreading by excising noncomplementary nucleotides prior to extension of the primer by polymerase action. The deoxynucleoside triphosphate analogs N2-(p-butylphenyl)deoxyguanosine triphosphate and N2-(p-butylphenyl)deoxyadenosine triphosphate are potent inhibitors of DNA polymerase alpha. Like calf thymus DNA polymerase delta, recently determined to have proofreading capability, DNA synthesis by the isolated Drosophila 182-kDa polymerase subunit was not inhibited by the two analogs. In contrast, DNA synthesis by the intact Drosophila polymerase-primase complex was inhibited greater than 95% by these analogs.
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PMID:Specificity of proofreading by the 3'----5' exonuclease of the DNA polymerase-primase of Drosophila melanogaster. 312 27

DNA repair synthesis following UV irradiation of confluent human fibroblasts has a biphasic time course with an early phase of rapid nucleotide incorporation and a late phase of much slower nucleotide incorporation. The biphasic nature of this curve suggests that two distinct DNA repair systems may be operative. Previous studies have specifically implicated DNA polymerase delta as the enzyme involved in DNA repair synthesis occurring immediately after UV damage. In this paper, we describe studies of DNA polymerase involvement in DNA repair synthesis in confluent human fibroblasts at late times after UV irradiation. Late UV-induced DNA repair synthesis in both intact and permeable cells was found to be inhibited by aphidicolin, indicating the involvement of one of the aphidicolin-sensitive DNA polymerases, alpha or delta. In permeable cells, the process was further analyzed by using the nucleotide analogue (butylphenyl)-2'-deoxyguanosine 5'-triphosphate, which inhibits DNA polymerase alpha several hundred times more strongly than it inhibits DNA polymerase delta. The (butylphenyl)-2'-deoxyguanosine 5'-triphosphate inhibition curve for late UV-induced repair synthesis was very similar to that for polymerase delta. It appears that repair synthesis at late times after UV irradiation, like repair synthesis at early times, is mediated by DNA polymerase delta.
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PMID:Involvement of DNA polymerase delta in DNA repair synthesis in human fibroblasts at late times after ultraviolet irradiation. 314 46

Porcine circovirus is the only mammalian DNA virus so far known to contain a single-stranded circular genome (Tischer et al. (1982) Nature 295, 64-66). Replication of its small viral DNA (1.76 kb) appears to be dependent on cellular enzymes expressed during S-phase of the cell cycle (Tischer et al. (1987) Arch. Virol. 96, 39-57). In this paper we have exploited the porcine circovirus genome to probe for in vitro initiation and elongation of DNA replication by different preparations of calf thymus DNA polymerase alpha and delta as well as by a partially purified preparation from pig thymus. The results indicated that three different purification fractions of calf thymus DNA polymerase alpha and one from pig thymus initiate DNA synthesis at several sites on the porcine circovirus DNA. It appears that the sites at which DNA primase synthesizes primers are not entirely random. Subsequent DNA elongation by a highly purified DNA polymerase alpha holoenzyme which had been isolated by the criterion of replicating single-stranded M13 DNA (Ottiger et al. (1987) Nucleic Acids Res. 15, 4789-4807) is very efficient. Complete conversion to the double-stranded form is obtained in less than 1 min. When the DNA synthesis by DNA polymerase alpha is blocked with the DNA polymerase alpha specific monoclonal antibody SJK 132-20 after initiation by DNA primase, DNA polymerase delta can efficiently replicate from the primers. This in vitro DNA replication system may be used in analogy to the bacteriophage systems in E. coli to study initiation and elongation of DNA replication.
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PMID:Replication of single-stranded porcine circovirus DNA by DNA polymerases alpha and delta. 320 61

When UV-irradiated cultured diploid human fibroblasts were permeabilized with Brij-58 then separated from soluble material by centrifugation, conservative DNA repair synthesis could be restored by a soluble factor obtained from the supernatant of similarly treated HeLa cells. Extensive purification of this factor yielded a 10.2 S, 220,000-dalton polypeptide with the DNA polymerase and 3'- to 5'-exonuclease activities reported for DNA polymerase delta II (Crute, J. J., Wahl, A. F., and Bambara, R. A. (1986) Biochemistry 25, 26-36). Monoclonal antibody to KB cell DNA polymerase alpha, while binding to HeLa DNA polymerase alpha, did not bind to the HeLa DNA polymerase delta. Moreover, at micromolar concentrations N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP) and 2-(p-n-butylanilino)-2'-deoxyadenosine 5'-triphosphate (BuAdATP) were potent inhibitors of DNA polymerase alpha, but did not inhibit the DNA polymerase delta. Neither purified DNA polymerase alpha nor beta could promote repair DNA synthesis in the permeabilized cells. Furthermore, under conditions which inhibited purified DNA polymerase alpha by greater than 90%, neither monoclonal antibodies to DNA polymerase alpha, BuPdGTP, nor BuAdATP was able to inhibit significantly the DNA repair synthesis mediated by the DNA polymerase delta. Thus, it appears that a major portion of DNA repair synthesis induced by UV irradiation might be catalyzed by DNA polymerase delta. When xeroderma pigmentosum human diploid fibroblasts were utilized, DNA repair synthesis dependent upon ultraviolet light could be restored by addition of both T4 endonuclease V and DNA polymerase delta, but not by addition of either one alone. This result suggests that cytosol-depleted permeabilized DNA repair-defective human fibroblasts and HeLa DNA polymerase delta might be exploited to provide a functional assay for purifying active DNA repair factors from DNA repair-proficient cells without a preknowledge of their function.
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PMID:DNA repair synthesis in human fibroblasts requires DNA polymerase delta. 333 6

The activities of DNA polymerases alpha and delta, in extracts from Chinese hamster ovary (CHO) cells, were assayed in order to determine whether these polymerases are regulated during the cell cycle. An exponential population of CHO cells was separated into enriched populations of G-1, S, and G-2/M phases of cell cycle by centrifugal elutriation. Total cell homogenates from each population were assayed for DNA polymerase activity by measuring labeled nucleotide incorporation into the exogenous templates oligo(dT).poly(dA) and DNase I activated calf thymus DNA. In these experiments, specific DNA polymerase inhibitors were added to assays of the cellular extracts to allow for the independent measurement of activities of DNA polymerases alpha and delta. Comparisons of total DNA polymerase activity from cellular extracts, sampled from each portion of the cell cycle, demonstrated no significant change with respect to the concentration of total protein. However, results indicate that the activity of DNA polymerase delta increases with respect to that of DNA polymerase alpha in the G-2/M portion of the cell cycle. This difference in relative activities of DNA polymerases alpha and delta suggests a coordinate regulation of a specific species of DNA polymerase during the cell cycle.
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PMID:Cell cycle dependent activities of DNA polymerases alpha and delta in Chinese hamster ovary cells. 342 9

The fidelity of DNA synthesis by calf thymus DNA polymerase delta (pol delta) in vitro has been determined using an M13lacZ alpha nonsense codon reversion assay. Pol delta is highly accurate, producing on average less than 1 single-base substitution error for each 10(6) nucleotides polymerized. This accuracy is 10- and 500-fold greater than that of DNA polymerases alpha and beta, respectively, in the same assay. Three observations suggest that this higher fidelity results in part from proofreading of misinserted bases by the 3' to 5' exonuclease associated with pol delta. First, the exonuclease efficiently excises terminally mismatched bases. Second, both terminal mismatch excision and the fidelity of DNA synthesis by pol delta are reduced with increasing concentration of deoxynucleoside triphosphates in the synthesis reaction. These effects result from increasing the rate of polymerization relative to the rate of exonucleolytic excision and are hallmarks of exonuclease proofreading. Third, both terminal mismatch excision and fidelity decrease upon addition to the reaction mixture of adenosine monophosphate, a compound known to selectively inhibit the exonuclease but not the polymerase activity of pol delta. These results suggest that 3' to 5' exonuclease-dependent proofreading enhances the fidelity of DNA synthesis by a mammalian DNA polymerase in vitro.
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PMID:Exonucleolytic proofreading by calf thymus DNA polymerase delta. 347 31


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