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:3.1.4.1 (phosphodiesterase)
18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Three major polypeptides are found in purified DNA polymerase alpha from rat liver: 160, 77 and 58 kDa. The electrophoretic analysis has identified polypeptide 160 kDa as the catalytically active subunit of DNA polymerase alpha. The other two polypeptides showed no DNA polymerase activity. Individual polypeptide p77 kDa purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to produce antibodies in rabbits. Immunoblot analysis indicated that the complex DNA polymerase alpha-3'-5'-exonuclease contained polypeptide p77 kDa. To elucidate the function of the p77 kDa protein we have prepared an immunoabsorbent column with antibodies against the p77 kDa polypeptide. The antibody column purified p77 kDa protein was homogeneous according to sodium dodecyl sulfate gel electrophoresis. The activity of alpha-polymerase was increased approximately 10-fold as a result of purification of DNA polymerase alpha from the p77 kDa protein. The in vitro experiments showed the identity of the p77 kDa polypeptide to endonuclease. It cleaved both single-stranded and double-stranded DNA. The function of endonuclease p77 kDA in complex with DNA polymerase alpha remains obscure.
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PMID:[Isolation and characteristics of endonuclease tightly bound to alpha-polymerase from the rat liver]. 284 24

DNA-dependent ATPase IV has been purified to near homogeneity from the Novikoff rat hepatoma. The enzyme is devoid of DNA polymerase, RNA polymerase, exonuclease, endonuclease, phosphomonoesterase, 3'- or 5'-phosphodiesterase, polynucleotide kinase, protein kinase, topoisomerase, helicase or DNA reannealing activities at a detection level of 10(-5) to 10(-7) relative to the ATPase activity. The enzyme is a monomer of Mr 110,000, has a sedimentation coefficient of 5.9 S, a Stokes radius of 40 A and a frictional coefficient of 1.32. In the presence of Mg2+ ion and a polynucleotide effector, ATPase IV hydrolyzes either ATP or dATP to the nucleoside diphosphate plus Pi. Other ribo- or deoxyribonucleoside triphosphates are not substrates. ATPase IV utilizes double-stranded DNA and single-stranded DNA as effector; however, it does not utilize poly(dT). The Km for dsDNA or ssDNA is 2.2 microM (nucleotide). A variety of ATP analogues were found to be competitive inhibitors of ATPase IV.
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PMID:Purification and enzymological characterization of DNA-dependent ATPase IV from the Novikoff hepatoma. 296 5

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 photoreaction of 8-methoxypsoralen (8-MOP) with DNA fragments of defined sequence was studied. We took advantage of the blockage by bulky adducts of the 3'-5'-exonuclease activity associated with the T4 DNA polymerase. The action of the exonuclease is stopped by biadducts as well as by monoadducts. The termination products were analyzed on sequencing gels. A strong sequence specificity was observed in the DNA photobinding of 8-MOP. The exonuclease terminates its digestion near thymine residues, mainly at potentially cross-linkable sites. There is an increasing reactivity of thymine residues in the order T less than TT much less than TTT in a GC environment. For thymine residues in cross-linkable sites, the reactivity follows the order AT much less than TA approximately TAT much less than ATA less than ATAT less than ATATAA. Repeated A-T sequences are hot spots for the photochemical reaction of 8-MOP with DNA. Both monoadducts and interstrand cross-links are formed preferentially in 5'-TpA sites. Our results highlight the role of the sequence and consequently of the conformation around a potential site in the photobinding of 8-MOP to DNA.
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PMID:Sequence context effects on 8-methoxypsoralen photobinding to defined DNA fragments. 365 86

Hematoporphyrin derivative (HPD) plus photoradiation caused the inactivation of DNA polymerases from calf thymus and R3230AC rat mammary tumor. Photosensitization of purified DNA polymerase-alpha as well as two forms of DNA polymerase-delta (I and II) from calf thymus were evaluated. Although all polymerase enzyme forms were inactivated at 70 micrograms HPD/ml, DNA polymerase-delta II was the most sensitive, displaying a 90% inactivation under conditions that did not cause significant inactivation of the other polymerase forms. Unlike DNA polymerase-alpha, the delta-forms have an associated 3'- to 5'-exonuclease activity. The exonuclease associated with DNA polymerase-delta II was uniquely sensitive to a low level of HPD and light exposure. DNA polymerase-delta II can be distinguished from other polymerase forms in cell extracts by its relative insensitivity to the polymerase inhibitor N2-(p-n-butylphenyl)deoxyadenosine 5'-triphosphate. In cytosols prepared from calf thymus and R3230AC rat mammary tumors, DNA polymerase-delta II was preferentially inhibited by HPD plus light. Furthermore, in experiments in which tumor-bearing rats were administered HPD prior to preparation of tumor cytosols, DNA polymerase-delta II was specifically inactivated by exposure to light. These results are discussed in view of their possible role in cancer therapy, and the potential use of HPD as a specific inhibitory agent of DNA polymerase-delta II is suggested.
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PMID:Inhibition of mammalian DNA polymerases by hematoporphyrin derivative and photoradiation. 394 Jan 88

The effects of Escherichia coli exonuclease I, exonuclease III, and deoxyribonucleic acid (DNA) polymerase on the biological activity of mature DNA from temperate Bacillus bacteriophage phi105 were investigated. Intact DNA loses infectivity rapidly upon exposure to exonuclease III. Although there is an overall decrease in marker rescue from exonuclease III-digested DNA, digestion preferentially affects markers at the end of the genetic map. This is taken to indicate a nonpermuted gene sequence in mature DNA. Incubation of mature DNA in the presence of exonuclease I or DNA polymerase has no effect on its biological activity. The possible structure of the ends of mature phi105 DNA is discussed. The rate of digestion of mature phi105 DNA by exonuclease III is only about 1/20 the rate of lambda DNA. Results of digestion of various DNA substrates by exonuclease III indicate that the enzyme distinguishes between different DNA terminal structures.
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PMID:Structure and biological activity of deoxyribonucleic acid from Bacillus bacteriophage phi 105: effects of Escherichia coli exonucleases. 432 11

1. DNA polymerase activity is present in both nuclear and supernatant fractions prepared from rapidly dividing L929 mouse cells. 2. Nuclear preparations are 2-5 times more active with added native DNA as template and the supernatant fractions show an equivalent preference for heat-denatured DNA. 3. Isolated nuclei can carry on limited DNA synthesis in the absence of added template but are stimulated five- to ten-fold by addition of 50mug of native DNA per assay. 4. DNA polymerase activity can be released from intact nuclei by ultrasonic treatment or by extraction with 1.5m-potassium chloride. 5. The activities in nuclear and supernatant fractions, with their preferred templates, respond similarly to changes in pH and Mg(2+) and K(+) concentrations. 6. Maximal enzyme activity is approached with 40mug of DNA per assay and activation of the DNA template by treatment with deoxyribonuclease does not decrease the amount of DNA required to reach saturation. 7. The nuclear enzyme, incubated with native DNA, is markedly inhibited by the addition of heat-denatured DNA to the assay. In contrast, the supernatant DNA polymerase activity on denatured templates is not affected by the presence of native DNA. 8. The nuclear enzyme exhibits high activity in the absence of one or more deoxyribonucleoside triphosphates but this is much diminished after partial purification of the enzyme by precipitation at pH5 and fractionation on Sephadex G-200 columns. 9. The (3)H-labelled DNA products formed by Sephadex-purified nuclear and supernatant fractions, with their preferred templates, were found to be resistant to treatment with exonuclease I. Alkali-denaturation of the (3)H-labelled DNA products rendered them susceptible to attack by exonuclease I. 10. Analysis of the products on alkaline sucrose density gradients suggests that the newly synthesized material may not be covalently bound to the original DNA template. 11. By using their preferred templates the specific activity of supernatant fractions varies markedly with the position of the cells in the cell-cycle, but the specific activity of nuclear fractions varies only slightly.
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PMID:Characteristics of deoxyribonucleic acid polymerase activity in nuclear and supernatant fractions of cultured mouse cells. 553 Nov 81

The inhibition of highly purified herpes simplex virus (HSV)-induced and host cell DNA polymerases by the triphosphate form of 9-(2-hydroxyethoxymethyl)guanine (acyclovir; acycloguanosine) was examined. Acyclovir triphosphate (acyclo-GTP) competitively inhibited the incorporation of dGMP into DNA, catalyzed by HSV DNA polymerase; apparent Km and Ki values of dGTP and acyclo-GTP were 0.15 microM and 0.003 microM, respectively. HeLa DNA polymerase alpha was also competitively inhibited; Km and Ki values of dGTP and acyclo-GTP were 1.2 microM and 0.18 microM, respectively. In contrast, HeLa DNA polymerase beta was insensitive to the analogue. The "limited" DNA synthesis observed when dGTP was omitted from HSV or alpha DNA polymerase reactions was inhibited by acyclo-GTP in a concentration-dependent manner. Prior incubation of activated DNA, acyclo-GTP, and DNA polymerase (alpha or HSV resulted in a marked decrease in the utilization of the primer-template in subsequent DNA polymerase reactions. This decreased ability of preincubated primer-templates to support DNA synthesis was dependent on acyclo-GTP, enzyme concentration, and the time of prior incubation. Acyclo-GMP-terminated DNA was found to inhibit HSV DNA polymerase-catalyzed DNA synthesis. Kinetic experiments with variable concentrations of activated DNA and fixed concentrations of acyclo-GMP-terminated DNA revealed a noncompetitive inhibition of HSV-1 DNA polymerase. The apparent Km of 3'-hydroxyl termini was 1.1 X 10(-7) M, the Kii and Kis of acyclo-GMP termini in activated DNA were 8.8 X 10(-8) M and 2.1 X 10(-9) M, respectively. Finally, 14C-labeled acyclo-GMP residues incorporated into activated DNA by HSV-1 DNA polymerase could not be excised by the polymerase-associated 3',5'-exonuclease activity.
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PMID:Inhibition of purified human and herpes simplex virus-induced DNA polymerases by 9-(2-hydroxyethoxymethyl)guanine triphosphate. Effects on primer-template function. 627 50

T4 DNA polymerase converts (Sp)-2'-deoxyadenosine 5'-O-(1-thio[1-18O2]triphosphate) to 2'-deoxyadenosine 5'-O-[18O]-phosphorothioate in the presence of poly(d(A-T).poly(d(A-T)) template-primer. Control experiments involving either omitting the poly(d(A-T)).poly(d(A-T) template-primer or employing the (Rp)-2'-deoxyadenosine 5'-O-(1-thiotriphosphate) diastereomer showed no reaction. It is assumed, therefore, that this conversion as in the P--O case involves incorporation of the thionucleotide into the poly(d(A-T)) followed by hydrolysis resulting from the 3' goes to 5'-exonuclease activity. The 2'-deoxyadenosine 5'-O-[18O] phosphorothioate was converted to (Sp)-2'-deoxyadenosine 5'-O-(1-thio[1-18O]triphosphate), with no change in the configuration at P alpha by using the coupled adenylate kinase-pyruvate kinase enzyme system. A 31P NMR spectrum of the product showed that the 18O was entirely in the nonbridging position, indicating an overall retention in the net turnover process (i.e. incorporation followed by excision). Since the incorporation process involves an inversion of configuration around the phosphorus (Romaniuk, P. J., and Eckstein, F. (1982) J. Biol. Chem. 257, 7684-7688), it must be inferred that the 3' goes to 5'-exonuclease activity of T4 polymerase proceeds with inversion of configuration at the phosphorus atom, most simply via a direct displacement mechanism. This finding represents the first example of phosphodiester hydrolysis catalyzed by an exonuclease that does not involve a covalent phosphoryl-enzyme intermediate (Knowles, J. R. (1980) Annu. Rev. Biochem. 49, 877-919).
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PMID:Template-prime-dependent turnover of (Sp)-dATP alpha S by T4 DNA polymerase. The stereochemistry of the associated 3' goes to 5'-exonuclease. 628 51

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


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