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)

It is well established that DNA replication and ultraviolet-induced DNA repair synthesis in mammalian cells are aphidicolin-sensitive and thus are mediated by one or both of the aphidicolin-sensitive DNA polymerases, alpha and/or delta. Recently, it has been shown that DNA polymerase delta is much more sensitive to inhibition by the nucleotide analogue 2',3'-dideoxythymidine 5'-triphosphate (ddTTP) than DNA polymerase alpha but is less sensitive than DNA polymerase beta [Wahl, A. F., Crute, J. J., Sabatino, R. D., Bodner, J. B., Marraccino, R. L., Harwell, L. W., Lord, E. M., & Bambara, R. A. (1986) Biochemistry 25, 7821-7827]. We find that DNA replication and ultraviolet-induced DNA repair synthesis in permeable human fibroblasts are also more sensitive to inhibition by ddTTP than polymerase alpha and less sensitive than polymerase beta. The Ki for ddTTP of replication is about 40 microM and that of repair synthesis is about 25 microM. These are both much less than the Ki of polymerase alpha (which is greater than 200 microM) but greater than the Ki of polymerase beta (which is less than 2 microM). These data suggest that DNA polymerase delta participates in DNA replication and ultraviolet-induced DNA repair synthesis in human cells.
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PMID:2',3'-Dideoxythymidine 5'-triphosphate inhibition of DNA replication and ultraviolet-induced DNA repair synthesis in human cells: evidence for involvement of DNA polymerase delta. 360 85

We have purified from Xenopus laevis ovaries a major DNA polymerase alpha species that lacked DNA primase activity. This primase-devoid DNA polymerase alpha species exhibited the same sensitivity as the DNA polymerase DNA primase alpha to BuAdATP and BuPdGTP, nucleotide analogs capable of distinguishing between DNA polymerase delta and DNA polymerase DNA primase alpha. The primase-devoid DNA polymerase alpha species also lacked significant nuclease activity indicative of the alpha-like (rather than delta-like) nature of the DNA polymerase. Using a poly(dT) template, the primase-devoid DNA polymerase alpha species elongated an oligo(rA10) primer up to 51-fold more effectively than an oligo(dA10) primer. In direct contrast, the DNA polymerase DNA primase alpha complex showed only a 4.6-fold preference for oligoribonucleotide primers at the same template/primer ratio. The catalytic differences between the two DNA polymerase alpha species were most dramatic at a template/primer ratio of 300. The primase-devoid DNA polymerase alpha species was found at high levels throughout oocyte and embryonic development. This suggests that the primase-devoid DNA polymerase alpha species could play a physiological role during DNA chain elongation in vivo, even if it is chemically related to DNA polymerase DNA primase alpha.
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PMID:Characterization of a stable, major DNA polymerase alpha species devoid of DNA primase activity. 369 91

In mammalian cells, both semiconservative DNA replication and the DNA repair patch synthesis induced by high doses of ultraviolet radiation are known to be inhibited by aphidicolin, indicating the involvement in these processes of one or both of the aphidicolin-sensitive DNA polymerases, alpha and/or delta. In this paper, N2-(p-n-butylphenyl)-2'-deoxyguanosine-5'-triphosphate, a strong inhibitor of polymerase alpha and a weak inhibitor of polymerase delta, is used to further characterize the DNA polymerase(s) involved in these two forms of nuclear DNA synthesis. In permeable human fibroblasts, DNA replication and ultraviolet-induced DNA repair synthesis are more resistant to the inhibitor than DNA polymerase alpha by factors of approximately 500 and 3000, respectively. These findings are most consistent with the involvement of DNA polymerase delta in these processes.
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PMID:DNA replication and UV-induced DNA repair synthesis in human fibroblasts are much less sensitive than DNA polymerase alpha to inhibition by butylphenyl-deoxyguanosine triphosphate. 376 98

The ontogeny of DNA polymerase activity in the neonatal rat heart was studied. The DNA polymerase activities in rat heart extracts were identified as DNA polymerase alpha and DNA polymerase delta activities by their purification and characterization, by the use of a specific inhibitor (BuAdATP), and by a specific monoclonal antibody against DNA polymerase alpha. Using these inhibitors, it was shown that the two activities declined in parallel during the maturation and terminal differentiation of the heart.
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PMID:Biochemical characterization and development of DNA polymerases alpha and delta in the neonatal rat heart. 381 35

The p-n-butylphenyl- and p-n-butylanilino- substituted analogs of dGTP and dATP, respectively, were tested as inhibitors of purified human placental DNA polymerases alpha and delta. It was observed that DNA polymerase alpha activity was potently inhibited by these analogs with I0.5 values as low as the nanomolar range, whereas DNA polymerase delta activity was poorly inhibited, with I0.5 values of ca. 100 micromolar. These results argue for a distinct identity of these two enzymes, and demonstrate the usefulness of these analogs as probes of DNA polymerase structures. In addition, these analogs provide a rapid method for the discrimination of the two enzyme activities and a means for the selective assay of DNA polymerase delta. Aphidicolin inhibited both DNA polymerases.
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PMID:Differential inhibition of human placental DNA polymerases delta and alpha by BuPdGTP and BuAdATP. 393 20

The effects of dimethylsulfoxide on the activities of purified human placental DNA polymerase alpha and DNA polymerase delta were examined. DNA polymerase alpha was inhibited by dimethylsulfoxide, whereas DNA polymerase delta was significantly activated, by as much as 6-fold. Kinetic data show that the effect of dimethylsulfoxide on DNA polymerase delta activity was due to a reduction in the apparent Km for its substrate, dTTP. This novel finding of the differential effects of dimethylsulfoxide on the activities of polymerases alpha and delta may be useful in their identification and differential assay.
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PMID:Differential effects of dimethylsulfoxide on the activities of human DNA polymerases alpha and delta. 395 94

Two high molecular weight DNA polymerases, which we have designated delta I and delta II, have been purified from calf thymus tissue. Using Bio Rex-70, DEAE-Sephadex A-25, and DNA affinity resin chromatography followed by sucrose gradient sedimentation, we purified DNA polymerase delta I 1400-fold to a specific activity of 10 000 nmol of nucleotide incorporated h-1 mg-1, and DNA polymerase delta II was purified 4100-fold to a final specific activity of 30 000 nmol of nucleotide incorporated h-1 mg-1. The native molecular weights of DNA polymerase delta I and DNA polymerase delta II are 240 000 and 290 000, respectively. Both enzymes have similarities to other purified delta-polymerases previously reported in their ability to degrade single-stranded DNA in a 3' to 5' direction, affinity for an AMP-hexane-agarose matrix, high activity on poly(dA) X oligo(dT) template, and relative resistance to the polymerase alpha inhibitors N2-(p-n-butylphenyl)dATP and N2-(p-n-butylphenyl)dGTP. These two forms of DNA polymerase delta also share several common features with alpha-type DNA polymerases. Both calf DNA polymerase delta I and DNA polymerase delta II are similar to calf DNA polymerase alpha in molecular weight, are inhibited by the alpha-polymerase inhibitors N-ethylmaleimide and aphidicolin, contain an active DNA-dependent RNA polymerase or primase activity, display a similar extent of processive DNA synthesis, and are stimulated by millimolar concentrations of ATP. We propose that calf DNA polymerase delta I, which also has a template specificity essentially identical with that of calf DNA polymerase alpha, could be an exonuclease-containing form of a DNA replicative enzyme.
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PMID:Purification and characterization of two new high molecular weight forms of DNA polymerase delta. 395 90

DNA polymerase delta from rabbit bone marrow has an associated 3'-5'-exonuclease. Previous studies demonstrated a Stokes radius of 45.5 A by gel filtration and a sedimentation coefficient of 6.5 S by zone sedimentation. Thus, a molecular weight of 122000 and a frictional coefficient of 1.39 were calculated [Byrnes, J. J., & Black, V. L. (1978) Biochemistry 17, 4226-4231]. Several problems obstructed further purification and definition of DNA polymerase delta. The small amount of protein obtained limited further purification as the nonspecific loss of enzyme in subsequent procedures was excessive. Furthermore, the amount of protein recovered was insufficient for conventional analysis. These difficulties have been overcome, and DNA polymerase delta has been purified to apparent homogeneity. Under conditions of nondenaturing microgel electrophoresis, DNA polymerase b aggregates to molecular weight species of 300000 and higher. In situ assays for DNA polymerase and exonuclease in these gels generate concordant activity profiles. Upon sodium dodecyl sulfate gel electrophoresis, delta is a single polypeptide of 122000 apparent molecular weight. The DNA polymerase incorporates between 250000 and 300000 nmol of thymidine deoxyribonucleoside monophosphate (dTMP) into poly(dA)/oligo(dT) (mg of protein)-1 h-2 at 37 degrees C; the exonuclease simultaneously hydrolyzes 13% of the newly synthesized DNA. Aphidicolin, considered to be a specific inhibitor of DNA polymerase alpha, inhibits both the DNA polymerase and 3'-5'-exonuclease activities of delta. DNA polymerase alpha from rabbit bone marrow does not share a common subunit with delta. Therefore, aphidicolin binding is not specific for alpha, and conclusions based upon the supposition that it is must be reconsidered.
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PMID:DNA polymerase delta: one polypeptide, two activities. 628 2

3'-5' Exonucleases from various sources were found to toxicogenate 4-hydroxycyclophosphamide ("activated" cyclophosphamide) by splitting the oxazaphosphorinane ring and releasing an alkylating moiety and acrolein. Neither cyclophosphamide (CP) nor the deactivated metabolites of CP, 4-keto-CP and carboxyphosphamide nor 4-(S-ethanol)-sulfido-CP were attacked by 3'-5' exonucleases. DNA polymerases with proofreading activity, such as DNA polymerase I from E. coli or DNA polymerase delta from rabbit bone marrow, exhibited a tenfold higher specific activity with "activated" CP than "plain" 3'-5' phosphodiesterases such as snake venom phosphodiesterase or 3',5'cyclic AMP phosphodiesterase from bovine heart tissue. High levels of toxicogenating activity were estimated in peripheric human lymphocytes and tissues of lymphatic origin, suggesting that enzymatic toxicogenation plays a key role with respect to the cytotoxic specificity of "activated" CP.
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PMID:Enzymatic toxicogenation of "activated" cyclophosphamide by 3'-5' exonucleases. 630 Jan 34

DNA polymerase delta, the most recently described class of eukaryotic DNA polymerase, has been purified to apparent homogeneity from rabbit bone marrow. Unlike the previously known eukaryotic DNA polymerases, delta has a 3' to 5' exonuclease as an integral component of its 122 000 molecular weight, single polypeptide structure. Similar to the function with prokaryotic DNA polymerases, the 3' to 5' exonuclease assists DNA polymerase delta in maintaining the fidelity of DNA synthesis by excising misincorporated nucleotides. DNA polymerase delta and the longer known eukaryotic DNA polymerase alpha are similar in many features. Both are very sensitive to sulfhydryl inhibitors such as N-ethylmaliemide (NEM) and to the antibiotic aphidicolin. Such criteria distinguish alpha and delta from DNA polymerases beta and gamma. This has led to the conclusion that nuclear DNA replication, which is sensitive to NEM and aphidicolin, is carried out by DNA polymerase alpha. However, the similar sensitivity of delta to these reagents requires that the role of alpha and delta in nuclear DNA replication be further defined. In many features DNA polymerase delta is also similar to the viral induced DNA polymerases such as the Herpes simplex virus DNA polymerases which also have associated 3' to 5' exonuclease. Understanding of DNA synthesis and the mechanism of DNA replication fidelity in mammalian cells depends upon a further understanding of both DNA polymerases alpha and delta and the nature of the relationship they have to each other.
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PMID:Structural and functional properties of DNA polymerase delta from rabbit bone marrow. 633 May 22


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