E F Baril

University of Nebraska Medical Center, Omaha, Nebraska, United States

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Publications (41)238.6 Total impact

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    ABSTRACT: Calcium and its receptor protein calmodulin function in the regulation of proliferation of mammalian cells. A 68 kDa calmodulin-specific binding protein was shown previously to be associated with growth factor-dependent progression of a variety of mammalian cells from G1 to S phase and to stimulate DNA synthesis in permeabilized hematopoietic progenitor cells. In this report we show that the 68 kDa calmodulin-specific binding protein in HeLa cells is tightly associated with the DNA polymerase alpha-primase component of the 21S complex of enzymes for DNA synthesis. The 68 kDa calmodulin-binding protein and the DNA polymerase alpha-primase complex cofractionate during Q-Sepharose chromatography to isolate the 21S enzyme complex, native and denatured DNA-cellulose to dissociate the 21S complex, and DEAE-Bio-Gel chromatography to isolate the multiprotein DNA polymerase alpha-primase complex. The 68 kDa calmodulin-specific binding protein and DNA polymerase alpha also bind and coelute during affinity chromatography on calmodulin-agarose. They also coprecipitate with C10-agarose-linked monoclonal antibody SJK 132-20 to human DNA polymerase alpha. The tight association of the 68 kDa calmodulin-binding protein to the DNA polymerase alpha-primase complex supports a function for this protein in the regulation of DNA synthesis in vivo.
    No preview · Article · Apr 1995 · Biochemistry
  • Qiu Ping Cao · Sidney Pitt · John Leszyk · Earl F. Baril
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    ABSTRACT: A 150-kDa DNA-dependent ATPase composed of 83/68-kDa subunits was previously reported to cofractionate with a 21S complex of enzymes for DNA synthesis from HeLa cells (Vishwanatha, J. K., & Baril, E. F. (1990) Biochemistry 29, 8753-8759). The DNA-dependent ATPase was purified to electrophoretic homogeneity from a HeLa cell homogenate by a modified procedure that involves subcellular fractionation, poly(ethylene-glycol) precipitation of the combined nuclear extract/cytosol, and chromatography on Q-Sepharose and native and denatured DNA/celluloses followed by Mono-S fast protein liquid chromatography. The purified enzyme showed equimolar amounts of 83- and 68-kDa polypeptides following polyacrylamide gel electrophoresis under denaturing conditions. Sequence analysis of peptide fragments derived from the separated 83- and 68-kDa polypeptides showed 90-100% homology with the corresponding 80- and 70-kDa subunits of human Ku protein. Immunoblot analysis of the ATPase during the course of its purification and immunoprecipitation with antibodies to the 80- and 70-kDa subunits of human Ku protein confirmed the relationship of the 83- and 68-kDa polypeptides of the human DNA-dependent ATPase to the subunits of human Ku protein. Both the 83- and 68-kDa polypeptides are phosphorylated by a DNA-dependent protein kinase that cofractionates with the ATPase. The DNA-dependent ATPase activity is up regulated by phosphorylation.
    No preview · Article · Aug 1994 · Biochemistry
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    Congjun Li · John Goodchild · Earl F. Baril
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    ABSTRACT: Approximately 80% of the DNA ligase activity in HeLa cell extracts is associated with the 21 S enzyme complex that functions in simian virus 40 DNA replication In vitro (Malkas et al., Biochemistry 29, 6362 – 6374., 1990). The DNA ligase associated with the 21 S complex was purified extensively and its physical, enzymlc and immunological properties characterized as DNA ligase I. The association of DNA ligase I with the 21 S complex of enzymes for DNA synthesis provides evidence for the physiological function of this DNA ligase In DNA replication In human cells.
    Full-text · Article · Mar 1994 · Nucleic Acids Research
  • Congjun Li · Long‐Guang Cao · Yu‐Li Wang · Earl F. Baril
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    ABSTRACT: The 21 S complex of enzymes for DNA synthesis in the combined low salt nuclear extract-post microsomal supernatant from HeLa cells [Malkas et al. (1990) Biochemistry 29:6362-6374] was purified by poly (ethylene glycol) precipitation, Q-Sepharose chromatography, Mono Q Fast Protein Liquid Chromatography (FPLC), and velocity gradient centrifugation. The procedure gives purified enzyme complex at a yield of 45%. The 21 S enzyme complex remains intact and functional in the replication of simian virus 40 DNA throughout the purification. Sedimentation analysis showed that the 21 S enzyme complex exists in the crude HeLa cell extract and that simian virus 40 in vitro DNA replication activity in the cell extract resides exclusively with the 21 S complex. The results of enzyme and immunological analysis indicate that DNA polymerase alpha-primase, a 3',5' exonuclease, DNA ligase I, RNase H, and topoisomerase I are associated with the purified enzyme complex. Denaturing polyacrylamide gel electrophoresis of the purified enzyme complex showed the presence of about 30 polypeptides in the size range of 300 to 15 kDa. Immunofluorescent imaging analysis, with antibodies to DNA polymerase alpha,beta and DNA ligase I, showed that polymerase alpha and DNA ligase I are localized to granular-like foci within the nucleus during S-phase. In contrast, DNA polymerase beta, which is not associated with the 21 S complex, is diffusely distributed throughout the nucleoplasm.
    No preview · Article · Dec 1993 · Journal of Cellular Biochemistry
  • Jamboor K. Vishwanatha · Earl F. Baril
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    ABSTRACT: A single-stranded DNA-dependent ATPase that cofractionates during the early stages of purification of a multiprotein DNA polymerase alpha complex from HeLa cells has been purified to homogeneity. The ATPase is part of a 16S multienzyme DNA polymerase alpha complex that is fully active in SV40 DNA replication in vitro. The ATPase hydrolyzes ATP to ADP in a reaction that is completely dependent on the presence of DNA. DNA in single-stranded form is strongly preferred as a cofactor, and polydeoxynucleotides with adenine or thymidine residues are highly effective. Glycerol gradient sedimentation showed that the purified ATPase sedimented at an s20,w of 7 S, and polyacrylamide gel electrophoresis under denaturing conditions reveals two polypeptides with relative molecular weights of 83,000 and 68,000. Both of these polypeptides have purine nucleotide binding sites as revealed by photoaffinity cross-linking experiments. ATP binds to the two subunits more efficiently than GTP, and CTP or UTP does not cross-link with the two polypeptides. DNA synthesis catalyzed by purified HeLa cell DNA polymerase alpha-primase is stimulated in the presence of ATPase and ATP at an optimum concentration of 2 mM. Analysis of the DNA product by gel electrophoresis indicates that with poly(dT) but not phage M13 DNA as template the ATPase overcomes a lag and decreases the length of nascent DNA chains synthesized by the DNA polymerase alpha-primase complex.
    No preview · Article · Oct 1990 · Biochemistry
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    ABSTRACT: A sedimentable complex of enzymes for DNA synthesis was partially purified from the combined low-salt nuclear extract-postmicrosomal supernatant solution of HeLa cell homogenates by poly(ethylene glycol) precipitation in the presence of 2 M KCl, discontinuous gradient centrifugation, Q-Sepharose chromatography, and velocity gradient centrifugation. In addition to the previously described 640-kDa multiprotein DNA polymerase alpha-primase complex [Vishwanatha et al. (1986) J. Biol. Chem. 261, 6619-6628], the enzyme complex also has associated topoisomerase I, DNA-dependent ATPase, RNase H, DNA ligase, a simian virus 40 origin recognition, dA/dT sequence binding protein [Malkas & Baril (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 70-74], and proliferating cell nuclear antigen. Essentially all of the T antigen dependent simian virus 40 in vitro replication activity in the combined nuclear extract-postmicrosomal supernatant solution resides with the sedimentable complex of enzymes for DNA synthesis. Sedimentation analysis on a 10-35% glycerol gradient in the presence of 0.5 M KCl indicates that the enzyme complex is 21S. The associated enzymes for DNA synthesis and in vitro simian virus 40 replication activity cofractionate throughout the purification of the 21S complex. The DNA polymerase and in vitro simian virus 40 replication activities are both inhibited by monoclonal antibody (SJK 132-20) to human DNA polymerase alpha and by 5-10 microM butylphenyl-dGTP, indicating that the association of DNA polymerase alpha with the 21S enzyme complex is essential for the initiation of SV40 DNA replication in vitro.
    Full-text · Article · Jul 1990 · Biochemistry
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    L H Malkas · E F Baril
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    ABSTRACT: A DNA-binding protein has been identified that recognizes runs of deoxyadenines and/or deoxythymines (dA/dT sequences) and purified from a chromatographic fraction containing the multiprotein DNA polymerase alpha-primase complex of HeLa cells by successive steps of chromatography on oligo(dT)-cellulose and Q-Sepharose. Polyacrylamide gel electrophoresis of the purified dA/dT sequence-binding protein in the presence of NaDodSO4 showed a single protein band of 62 kDa. Nitrocellulose filter binding assays using homopolydeoxynucleotides indicated that the purified protein preferentially binds to dA/dT sequences in single-stranded or duplex DNAs. Gel mobility shift assays with a variety of DNAs showed that the purified protein specifically binds to a fragment of simian virus 40 DNA containing the minimal (core) origin for replication. The binding occurred in a protein-dependent manner and in the presence of a vast excess of competing DNAs lacking the simian virus replication origin. The origin binding was reduced, however, when DNA fragments from simian virus 40 deletion mutants containing deletions within the 17-base-pair A + T-rich tract in the core DNA replication origin were used in the assays. These results indicate that the dA/dT sequence-binding protein preferentially binds to the 17-base-pair A + T-rich tract and suggest a possible role for the protein in the initiation of DNA replication.
    Full-text · Article · Feb 1989 · Proceedings of the National Academy of Sciences
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    Full-text · Article · Jan 1988 · Cancer cells (Cold Spring Harbor, N.Y.: 1989)
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    RJ Hickey · LH Malkas · N Pedersen · C Li · EF Baril

    Full-text · Chapter · Jan 1988
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    J K Vishwanatha · E F Baril
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    ABSTRACT: DNA primase activity has been resolved from a purified DNA primase-polymerase alpha complex of HeLa cells by hydrophobic affinity chromatography on phenylSepharose followed by chromatography on hexylagarose. This procedure provides a good yield (55%) of DNA primase that is free from polymerase alpha. The free DNA primase activity was purified to near homogeneity and its properties characterized. Sodium dodecyl sulfate polyacrylamide gel electrophoretic analysis of the purified free DNA primase showed a major protein staining band of Mr 70,000. The native enzyme in velocity sedimentation has an S20'W of 5. DNA primase synthesizes RNA oligomers with single-stranded M-13 DNA, poly(dT) and poly(dC) templates that are elongated by the DNA polymerase alpha in a manner that has already been described for several purified eukaryotic DNA primase-polymerase alpha complexes. The purified free DNA primase activity is resistant to neutralizing anti-human DNA polymerase alpha antibodies, BuPdGTP and aphidicolin that specifically inhibit the free DNA polymerase alpha and also DNA polymerase alpha complexed with the primase. The free primase activity is more sensitive to monovalent salt concentrations and is more labile than polymerase alpha. Taken together these results indicate that the DNA primase and polymerase alpha activities of the DNA primase-polymerase alpha complex reside on separate polypeptides that associate tightly through hydrophobic interactions.
    Preview · Article · Dec 1986 · Nucleic Acids Research
  • Jamboor K. Vishwanatha · Earl F. Baril
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    ABSTRACT: DNA primase activlty has been resolved from a purified DNA primase-polymerase α complex of HeLa cells by hydrophobia affinity chromatography on phenylSepharose followed by chromatography on hexylagarose. This procedure provides a good yield (55%) of DNA primase that is free from polymerase α. The free DMA primase activity was purified to near homogeneity and its properties characterized. Sodium dodecyl sulfate polyaorylanide gel electrophoretio analysis of the purified free DNA primase showed a major protein staining band of Mr 70,000. The native enzyme in velcity sedimentation has an S20, W of 5. DNA primase synthesizes RNA ollgomers with single- stranded M-13 DNA, poly(dT) and poly(dC) templates that are elongated by the DNA polymerase α in a manner that has already been described for several purified eukaryotic DNA primase-polymerase α complexes. The purified free DNA prinase activity is resistant to neutralizing anti-human DNA polymerase α antibodies, BuPdGTP and aphidicolin that specifically inhibit the free DNA polymerase α and also DNA polymerase α complexed with the primase. The free primase activity is more sensitive to monovalent salt concentrations and is more labile than polymerase α. Taken together these results indicate that the DNA primase and polymerase α activities of the DNA primase-polymerase α complex reside on separate polypeptides that associate tightly through hydrophobio Interactions.
    No preview · Article · Nov 1986 · Nucleic Acids Research
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    ABSTRACT: Synthesis of (p)ppRNA-DNA chains by purified HeLa cell DNA primase-DNA polymerase α (pol α-primase) was compared with those synthesized by a multiprotein form of DNA polymerase α (pol α2) using unique single-stranded DNA templates containing the origin of replication for simian virus 40 (SV40) DNA. The nucleotide locations of 33 initiation sites were identified by mapping G*pppN-RNA-DNA chains and identifying their 5'-terminal ribo-nucleotide. Pol α2 strongly preferred initiation sites that began with ATP rather than GTP, thus frequently preferring different initiation sites than pol α-primase, depending on the template examined. The initiation sites selected in vitro, however, did not correspond to the sites used during SV40 DNA replication in vitro. Pol α2 had the greatest effect on RNA primer size, typically synthesizing primers 1–5 nucleotides long, while pol α-primase synthesized primers 6–8 nucleotides long. These differences were observed even at individual initiation sites. Thus, the multiprotein form of DNA primase-DNA polymerase a affects selection of initiation sites, the frequency at which the sites are chosen, and length of RNA primers.
    Preview · Article · Oct 1986 · Nucleic Acids Research
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    W Skarnes · P Bonin · E Baril
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    ABSTRACT: The DNase that is associated with a multiprotein form of HeLa cell DNA polymerase alpha (polymerase alpha 2) has two distinct exonuclease activities: the major activity initiates hydrolysis from the 3' terminus and the other from the 5' terminus of single-stranded DNA. The two exonuclease activities show identical rates of thermal inactivation and coincidental migration during chromatofocusing, glycerol gradient centrifugation, and nondenaturing polyacrylamide gel electrophoresis of the DNase. Moreover, the purified DNase shows a single protein band of Mr 69,000 following nondenaturing polyacrylamide and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 3'----5' exonuclease activity hydrolyzes only single-stranded DNA substrates and the products are 5' mononucleotides. This activity recognizes and excizes mismatched bases at the 3' terminus of double-stranded DNA substrates. The 3'----5' exonuclease does not hydrolyze 3' phosphoryl terminated single-stranded DNA substrates. The 5'----3' exonuclease activity also only hydrolyzes single-stranded DNA substrates. The rate of hydrolysis, however is only about 1/25th the rate of the 3'----5' exonuclease. This exonuclease activity requires a 5' single-stranded terminus in order to initiate hydrolysis and does not proceed into double-stranded regions. The products of hydrolysis by 5'----3' exonuclease are also 5' nucleoside monophosphates.
    Preview · Article · Jun 1986 · Journal of Biological Chemistry
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    ABSTRACT: The majority of the DNA polymerase alpha activity in HeLa cells has been isolated and purified as a multiprotein Mr 640,000 form. The multiprotein form of DNA polymerase alpha corresponds to DNA polymerase alpha 2 that was previously reported by us (Lamothe, P., Baril, B., Chi, A., Lee, L., and Baril, E. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 4723-4727). The highly purified DNA polymerase alpha 2 has in addition to DNA polymerase alpha-associated DNase, primase, and diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A)binding activities and accessory primer recognition proteins C1 and C2. The DNA polymerase alpha and associated activities increase coordinately during the G1/S-phase transition of the cell cycle. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the electrophoretically homogeneous DNA polymerase alpha shows that it is composed of at least eight polypeptides in the molecular weight range of 180,000-15,000. Hydrophobic chromatography on butyl-agarose resolves the DNase and Ap4A-binding protein from a complex of DNA polymerase alpha, primase, and the primer recognition proteins C1 and C2. Hydrophobic chromatography of the latter complex on phenyl-Sepharose resolves the C1 protein from a DNA polymerase alpha-C2 protein-primase complex. Phosphocellulose chromatography of the DNA polymerase-primase-C2 protein complex resolves the C2 protein from a complex of DNA polymerase alpha-primase.
    Preview · Article · Jun 1986 · Journal of Biological Chemistry
  • E F Baril · S A Coughlin · P C Zamecnik
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    ABSTRACT: The proposal that Ap4A acts as an inducer of DNA replication is based primarily on two pieces of evidence (7). The intracellular levels of Ap4A increase ten- to 1000-fold as cells progress into S phase and the introduction of Ap4A into nonproliferating cells stimulated DNA synthesis. There is also some additional suggestive evidence such as the binding of Ap4A to a protein that is associated with multiprotein forms of the replicative DNA polymerase alpha and the ability of this enzyme to use Ap4A as a primer for DNA synthesis in vitro with single-stranded DNA templates. These observations have stimulated interest in the cellular metabolism of Ap4A. This is well since there is a great need for additional experimentation in order to clearly establish Ap4A as an inducer of DNA replication. Microinjection experiments of Ap4A into quiescent cells are needed in order to ascertain if Ap4A will stimulate DNA replication and possibly cell division in intact cells. Studies of the effects of nonhydrolyzable analogs of Ap4A on DNA replication in intact quiescent cells could also prove valuable. Although Ap4A can function as a primer for in vitro DNA synthesis by DNA polymerase alpha this may not be relevant in regard to its in vivo role in DNA replication. Ap4A in vivo could interact with key protein(s) in DNA replication and in this way act as an effector molecule in the initiation of DNA replication. In this regard the interaction of Ap4A with a protein associated with a multiprotein form of DNA polymerase alpha isolated from S-phase cells is of interest. More experiments are required to determine if there is a specific target protein(s) for Ap4A in vivo and what its role in DNA replication is. The cofractionation of tryptophanyl-tRNA synthetase with the replicative DNA polymerase alpha from animal and plant cells is of interest. The DNA polymerase alpha from synchronized animal cells also interacted with Ap4A. Although the plant cell alpha-like DNA polymerase did not interact with Ap4A this DNA polymerase was not a multiprotein form of polymerase alpha and the synchrony of the wheat germ embryos was not known. A possible tie between protein-synthesizing systems and the regulation of proteins involved in DNA replication may exist. The requirement of protein synthesis for the initiation of DNA replication has long been known. Also, it is well established that many temperature-sensitive mutants for tRNA synthetases are also DNA-synthesizing mutants. More investigation in this area may be warranted.(ABSTRACT TRUNCATED AT 400 WORDS)
    No preview · Article · Feb 1985 · Cancer Investigation
  • Earl F. Baril · Susan A. Coughlin · Paul C. Zamecnik

    No preview · Article · Jan 1985 · Cancer Investigation
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    E Baril · P Bonin · D Burstein · K Mara · P Zamecnik
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    ABSTRACT: A diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) binding subunit has been resolved from a high molecular weight (640,000) multiprotein form of DNA polymerase alpha [deoxynucleoside triphosphate:DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7] from HeLa cells [DNA polymerase alpha 2 of Lamothe, P., Baril, B., Chi, A., Lee, L. & Baril, E. (1981) Proc. Natl. Acad. Sci. USA 78, 4723-4727]. The Ap4A binding activity copurifies with the DNA polymerizing activity during the course of purification. Hydrophobic chromatography on butylagarose resolves the Ap4A binding activity from the DNA polymerase. The Ap4A binding activity is protein in nature since the binding of Ap4A is abolished by treatment of the isolated binding activity with proteinase K but is insensitive to treatment with DNase or RNase. The molecular weight of the Ap4A binding protein, as determined by polyacrylamide gel electrophoresis under nondenaturing conditions or by NaDodSO4/polyacrylamide gel electrophoresis after photoaffinity labeling of the protein with [32P]Ap4A is 92,000 or 47,000. The binding activity of this protein is highly specific for Ap4A.
    Preview · Article · Aug 1983 · Proceedings of the National Academy of Sciences
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    ABSTRACT: Terminal deoxynucleotidyl transferase (TdT) is a unique DNA synthetic enzyme whose expression in normal cells is restricted to subsets of primitive lymphocytes, and among neoplastic cells is found only in the blast cells of some forms of acute leukemia and diffuse lymphoma [1–5]. This polymerase has achieved increasing attention over the last several years from both basic biologists and physicians caring for patients with malignant hematologic disease. For biologists it has emerged as a useful biochemical marker for subsets of pre-B and pre-T cells, and has played an important role in studies aimed at dissecting the ontogeny of the lymphoid system [6, 7]. In clinical medicine, blast cell TdT assays have been shown to be useful in the subclassification of acute leukemias and diffuse lymphomas, and TdT status has been used to assign patients to therapeutically meaningful categories [8, 9].
    Preview · Article · Feb 1983 · Haematology and blood transfusion
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    G E Wright · E F Baril · V M Brown · N C Brown
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    ABSTRACT: The 2-amino substituted derivatives of guanine, N2-(p-n-butylphenyl)guanine (BuPG) and N2-(3',4'-trimethylenephenyl) guanine (TMPG), were synthesized and found to selectively inhibit, respectively, HeLa cell DNA polymerase alpha (po1 alpha) and B. subtilis DNA polymerase III (po1 III). Both purines, like their corresponding uracil analogs, BuAu and TMAU (2,9), were specifically competitive with dGTP in their inhibitory action on their target polymerases. BuPG, the pol alpha-specific purine, was also toxic for HeLa cells in vivo, selectively inhibiting DNA synthesis. These N2-substituted purines, in contrast to the 6-substituted uracils, provide a structural basis for the synthesis of nucleosides and nucleotides with considerable potential as probes for the analysis of the structure of specific replicative DNA polymerases and their function in cellular DNA metabolism.
    Preview · Article · Aug 1982 · Nucleic Acids Research
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    ABSTRACT: The 2-amino substituted derivatives of guanine. N2-(p-n-butylphenyl)guanine (BuPG) and Nh(3',4'-trimethylenephenyl) guanine (TMPG); wae synthesized and found to selectively inhibit, respectively, HeLa cell DNA polymerase alpha (pol α) and B. subtilis DNA polymerase III ( pol III). Both purines, like their corresponding uracil analogs, BuAU and TMAU (2.9), were specifically competitive with dGTP in their inhibitory action on their target polymerases. BuPG, the pola α-specific purine, was also toxic for HeLa cells in vivo, selectively inhibiting DNA synthesis. These N2-substituted purines, in contrast to the 6-substituted uracils, provide astructural basis for the synthesis of nucleosides and nucleotides with considerable potential as probes for the analysis of the structure of specific replicative DNA polyrnerases and their function incellular DNA metabolism.
    Preview · Article · Jul 1982 · Nucleic Acids Research

Publication Stats

1k Citations
238.60 Total Impact Points

Institutions

  • 1990
    • University of Nebraska Medical Center
      • Department of Biochemistry
      Omaha, Nebraska, United States
  • 1986
    • Harvard University
      • Department of Chemistry and Chemical Biology
      Cambridge, Massachusetts, United States
  • 1982
    • University of Massachusetts Medical School
      • Department of Cell Biology
      Worcester, Massachusetts, United States
  • 1970-1973
    • Duke University Medical Center
      • Department of Medicine
      Durham, North Carolina, United States