Modified nucleotide polymers as inhibitors of DNA polymerases.

We have compared the relative inhibitory activity of poly (A) with its analogues poly N6-isopentenyl adenylic acid (poly(i6 A)) and poly N6-benzyl adenylic acid (poly(bzl6A)), and of poly (U) with its analogue poly 2'-fluoro-2'-deoxyuridylic acid (poly(dUfl)), against DNA polymerase, alpha, beta and gamma and terminal deoxynucleotidyl transferase from human cells and two oncorna virus DNA polymerases. Although poly (A) and its analogues were equally inhibitory against endogenous RNA-directed DNA polymerases of murine and feline leukemia viruses, the analogues in contrast to poly (A) were strongly inhibitory against all four cellular enzymes. Poly (dUfl), on the other hand, was up to 100-fold more potent than poly (U) against both viral and cellular enzymes. Since poly (U) at 100 mug/ml and poly (dUfl) at 1 mug/ml had no effect on terminal deoxynucleotidyl transferase while inhibiting other enzymes by 80--100 per cent these polymers could be useful in the characterization and assay of terminal deoxynucleotidyl transferase. In addition, the polymers such as poly (igA) and poly (bzl5A) which were strongly inhibitory to all cellular enzymes, could be useful in cancer chemotherapy if taken up preferentially by the malignant calls due to their high pinocytic activity. The results also demonstrate potential for large variation in inhibitory activity of polyribonucleotides as related to their chemical composition.     

Comparative studies on polyguanylate polymerase and polyadenylate polymerase activities in the DNA-dependent RNA polymerase I fraction from cauliflower.

The properties of poly(G) polymerase and poly(A) polymerase activities in the DNA-dependent RNA polymerase [nucleosidetriphosphate: RNA nucleotidyltransferase EC 2.7.7.6] I fraction from cauliflower (Brassica oleracea var. botrytis) were comparatively investigated. The pH optimum, the effect of ionic strength, the effect of substrate concentration on the rate of synthesis, the effect of divalent metal ion concentration, and the time course of synthesis at different temperatures were all different for the three polymerase activities. The enzyme fraction preferentially utilized denatured DNA. Synthetic poly(C) and poly(U) were more effectively utillized for the synthesis of polyguanylate and polyadenylate, respectively. Further, it was found that poly(G) and poly(A) formed in vitro by the enzyme fraction had chain length of 25-28 and 84-89 nucleotides, respectively, and that poly (adenylate-gluanylate) chain was hardly formed when ATP and GTP were added together as substrates in the same reaction medium.     

Poly(ADP-ribose) polymerase from Ehrlich ascites tumor cells. Properties of the purified polymerase.

Poly(ADP-ribose) polymerase from Ehrlich ascites tumor cells, partially purified by chromatography on DNA-agarose, was obtained as a more than 80% homogeneous preparation by isoelectric focusing in a sucrose gradient. The polymerase activity was shown to be associated with the major protein in the preparation. Results obtained by electrophoresis in the presence of sodium dodecyl-sulfate indicated that poly(ADP-ribose) polymerase consists of a polypeptide chain with a molecular weight of 130 000. Ultracentrifugation at non-denaturating conditions indicated that the active enzyme may be an oligomeric form of this polypeptide chain. The isoelectric point of the polymerase was 9.40. The effects of various additions to the assay mixture on the synthesis of poly(ADP-ribose) as well as some kinetic data, are given. It is shown that poly(ADP-ribose) is a highly efficient inhibitor of its own synthesis, and results are presented which suggest that the well-known stimulatory effect of DNA on the synthesis is due to reduction of this inhibitory effect of the product.     

Different effects of tert-butylhydroperoxide-induced peroxynitrite-dependent and -independent DNA single-strand breakage on PC12 cell poly(ADP-ribose) polymerase activity.

The short-chain lipid hydroperoxide analogue tert-butylhydroperoxide induces peroxynitrite-dependent and -independent DNA single strand breakage in PC12 cells. U937 cells that do not express constitutive nitric oxide synthase respond to tert-butylhydroperoxide treatment with peroxynitrite-independent DNA cleavage. Under experimental conditions leading to equivalent strand break frequencies, the analysis of poly(ADP-ribose) polymerase activity showed an increase in PC12 cells but not in U937 cells. The enhanced poly(ADP-ribose) polymerase activity observed in PC12 cells was paralleled by a significant decline in NAD+ content and both events were prevented by treatments suppressing formation of peroxynitrite. Although DNA breaks were rejoined at similar rates in the two cell lines, an inhibitor of poly(ADP-ribose) polymerase delayed DNA repair in PC12 cells but had hardly any effect in U937 cells. The results obtained using the latter cell type were confirmed with an additional cell line (Chinese hamster ovary cells) that does not express nitric oxide synthase. Collectively, our data suggest that tert-butylhydroperoxide-induced peroxynitrite-independent DNA strand scission is far less effective than the DNA cleavage generated by endogenous peroxynitrite in stimulating the activity of poly(ADP-ribose) polymerase.     

Poly(A) Polymerase from Vaccinia Virus-Infected Cells I. Partial Purification and Characterization

Poly(A) polymerase activity is induced during vaccinia virus infection of HeLa cells. The enzyme is maximally induced at 3.5 h postinfection. Partial purification frees the preparation of RNase activity and RNA polymerase activity. ATP is the substrate for poly(A) synthesis. A small amount of poly(A) is produced from added adenosine diphosphate due to the production of ATP by an adenylate kinase present in the preparation. The incorporation of ATP into poly(A) is dependent on divalent cations (Mg²⁺ or Mn²⁺) and is not inhibited by UTP, CTP, or GTP. Poly(U) stimulates ATP incorporation; poly(A) and poly(C) have little effect on ATP incorporation, and poly(dT) is extremely inhibitory. RNA prepared from HeLa cells and from the partially purified poly(A) polymerase (the enzyme preparation contains endogenous RNA [Brakel and Kates]) stimulates ATP incorporation by poly(A) polymerase which was subjected to DEAE-cellulose chromatography. RNase's, pancreatic and T₁, inhibit the production of poly(A). DNase has little effect. Poly(U) is able to stimulate poly(A) production in the presence of T₁ RNase.     

Differential susceptibilities of DNA polymerases-alpha and -beta to polyanions.

The effects of various polyanions including synthetic polynucleotides on DNApolymerases-alpha and -beta from blastulae of the sea urchin Hemicentrotus pulcherrimus and HeLa cells were studied. Only DNA polymerase-alpha was inhibited by polyanions, such as polyvinyl sufate, dextran sulfate, heparin, poly(G), poly(I), poly(U) and poly(ADP-Rib). Of the various polynucleotides tested, poly(G) and poly(I) were the strongest inhibitors. Kinetic studies showed that the Ki value for poly(G) was 0.3 microgram/ml and that poly(G) had 20-fold higher affinity than activated DNA for the template-primer site of DNA polymerase-alpha. Poly(U) and poly(ADP-Rib) were also inhibitory, but they were one hundredth as inhibitory as poly(G) or poly(I). Poly(A), poly(C), poly(A).poly(U) AND POLY(I).poly(C) were not inhibitory to DNA polymerase-alpha. In contrast, DNA olymerase-beta was not affected at all by these polyanions under the same conditions.     

Antibacterial nitroacridine, Nitroakridin 3582: binding to nucleic acids in vitro and effects on selected cell-free model systems of macromolecular biosynthesis

Nitroakridin 3582 (NA) formed complexes with native deoxyribonucleic acid (DNA) and with transfer ribonucleic acid (tRNA) species from Escherichia coli. Spectrophotometric titrations of NA with these nucleic acids produced numerical results from which nonlinear adsorption isotherms were derived. These curves indicated the existence of more than one class of binding sites on the polymers to which NA was bound by more than one process. The stoichiometry of strong binding of NA to double helical DNA was in agreement with a conventional value (1 ligand molecule per 4.2 component nucleotides) for complete intercalation binding. NA inhibited the DNA-dependent DNA polymerase I and RNA polymerase reactions, the first strongly and the second appreciably. These inhibitions corresponded to the extents to which NA inhibits DNA and RNA biosyntheses in vivo. Evidently, NA interferes with the template function of DNA. The drug also inhibited the polymerization of phenylalanine in a cell-free E. coli ribosome-polyuridylic acid [poly (U)] system. The effect paralleled an inhibition of the poly (U)-directed binding of phenylalanyl tRNA to ribosomes. Ethidium bromide acted similarly. The antimalarial drug, chloroquine, stimulated polyphenylalanine synthesis, apparently as a result of stimulating the poly (U)-directed binding of phenylalanyl tRNA to ribosomes.     

Non-snRNP U1A levels decrease during mammalian B-cell differentiation and release the IgM secretory poly(A) site from repression

A regulated shift from the production of membrane to secretory forms of Immunoglobulin M (IgM) mRNA occurs during B cell differentiation due to the activation of an upstream secretory poly(A) site. U1A plays a key role in inhibiting the expression of the secretory poly(A) site by inhibiting both cleavage at the poly(A) site and subsequent poly(A) tail addition. However, how the inhibitory effect of U1A is alleviated in differentiated cells, which express the secretory poly(A) site, is not known. Using B cell lines representing different stages of B cell differentiation, we show that the amount of U1A available to inhibit the secretory poly(A) site is reduced in differentiated cells. Undifferentiated B cells have more total U1A than differentiated cells and a greater proportion of this is not associated with the U1snRNP. We show that this is available to inhibit poly(A) addition at the secretory poly(A) site using cold competitor RNA oligos to de-repress poly(A) addition in nuclear extracts from the respective cell lines. In addition, endogenous non-snRNP associated U1A-immunopurified from the different cell lines-inhibits poly(A) polymerase activity proportional to U1A recovered, suggesting that available U1A level alone is responsible for changes in its inhibitory effect at the secretory IgM poly (A) site.     

Inhibition of DNA polymerase alpha, DNA polymerase beta, terminal deoxynucleotidyl transferase, and DNA ligase II by poly(ADP-ribosyl)ation reaction in vitro

Incubation of DNA polymerase alpha, DNA polymerase beta, terminal deoxynucleotidyl transferase, or DNA ligase II in a reconstituted poly(ADP-ribosyl)ating enzyme system markedly suppressed the activity of these enzymes. Components required for poly(ADP-ribose) synthesis including poly(ADP-ribose) polymerase, NAD+, DNA, and Mg2+ were all essential for the observed suppression. Purified poly(ADP-ribose) itself, however, was slightly inhibitory to all of these enzymes. Furthermore, the suppressed activities of DNA polymerase alpha, DNA polymerase beta, and terminal deoxynucleotidyl transferase were largely restored (3 to 4-fold stimulation was observed) by a mild alkaline treatment, a procedure known to hydrolyze alkaline-labile ester linkage between poly(ADP-ribose) and an acceptor protein. All of these results strongly suggest that the four nuclear enzymes were inhibited as a result of poly(ADP-ribosyl)ation of either the enzyme molecule itself or some regulatory proteins of these enzymes.     

Template specificities of Aclacinomycin B on the inhibition of DNA-dependent RNA synthesis in vitro

Summary The effect of Aclacinomycin B (ACM-B), an anthracycline antitumor antibiotic, on the DNA-dependent RNA synthesis using single- and double-stranded DNAs of known base content and sequence is studied. The data show that ACM-B effectively inhibits the double-stranded DNA-directed RNA synthesis with a preference of poly[d(A-T)] > poly[d(G-C)] > poly[d(I-C)]. In contrast, it has no inhibitory effect on the template function of single-stranded DNA (e.g. poly dA, poly dT, and poly dC). These results suggest that the mechanism of ACM-13 inhibition, like other anthracycline antibiotics, is by intercalation. In addition to the base specificity, there are also dramatic differences in inhibition depending on the base sequence in the DNA template. Thus, ACM-13 preferentially inhibits the alternating double-stranded copolymers over the double-stranded homopolymers; e.g. poly [d(A-T)] is inhibited to a greater extent than poly dA · poly dT and poly [d(G-C)] is inhibited more than poly dG · poly dC. Since the inhibition by ACM-13 can be totally abolished when assayed in excess amount of DNA, this result suggests that ACM-B inhibition of RNA synthesis is solely on the DNA template (which is in support of the intercalation model), and has ruled out the possibility that ACM-B may also exert an inhibitory effect on the activity of RNA polymerase per se.     

Dideoxynucleoside triphosphate-sensitive DNA polymerase from rice is involved in base excision repair and immunologically similar to mammalian DNA pol beta

A single polypeptide with ddNTP-sensitive DNA polymerase activity was purified to near homogeneity from the shoot tips of rice seedlings and analysis of the preparations by SDS-PAGE followed by silver staining showed a polypeptide of 67 kDa size. The DNA polymerase activity was found to be inhibitory by ddNTP in both in vitro DNA polymerase activity assay and activity gel analysis. Aphidicolin, an inhibitor of other types of DNA polymerases, had no effect on plant enzyme. The 67 kDa rice DNA polymerase was found to be recognized by the polyclonal antibody (purified IgG) made against rat DNA polymerase beta (pol beta) both in solution and also on Western blot. The recognition was found to be very specific as the activity of Klenow enzyme was unaffected by the antibody. The ability of rice nuclear extract to correct G:U mismatch of oligo-duplex was observed when oligo-duplex with 32P-labeled lower strand containing U (at 22nd position) was used as substrate. Differential appearance of bands at 21-mer, 22-mer, and 51-mer position in presence of dCTP was visible only with G:U mismatch oligo-duplex, but not with G:C oligo-duplex. While ddCTP or polyclonal antibody against rat-DNA pol beta inhibits base excision repair (BER), aphidicolin had no effect. These results for the first time clearly demonstrate the ability of rice nuclear extract to run BER and the involvement of ddNTP-sensitive pol beta type DNA polymerase. Immunological similarity of the ddNTP-sensitive DNA polymerase beta of rice and rat and its involvement in BER revealed the conservation of structure and function of ddNTP-sensitive DNA pol beta in plant and animal.     

Enrichment of specific genes from genomic DNA or from clone library DNA, using R-looping

We have developed a procedure for enriching DNA for specific sequences that is based on R-looping (Thomas et al., 1976). R-loops are formed with the DNA using mRNAs containing the sequence of interest and then isolated on poly(U)-sepharose via the poly(A) tail of the mRNA. Model experiments showed that plasmid DNA containing a cDNA copy of an immunoglobulin kappa chain mRNA could be selectively retrieved using this procedure. Approx. 5-10% of the kappa sequences in mouse embryo DNA could be recovered by R-looping, while non-specific binding of mouse DNA to the poly(U)-sepharose column was 0.03-0.04%. This represents a 100-200-fold enrichment of mouse genomic kappa sequences. We have also used the procedure to rapidly screen a mouse clone library for immunoglobulin heavy chain genes. DNA from the clone library was enriched 100-200-fold using immunoglobulin heavy chain mRNAs, and the enriched DNA repackaged in vitro to recover the phage.     

Isolation of an RNA-directed RNA polymerase-specific cDNA clone from tomato.

A 3600-bp RNA-directed RNA polymerase (RdRP)-specific cDNA comprising an open reading frame (ORF) of 1114 amino acids was isolated from tomato. The putative protein encoded by this ORF does not share homology with any characterized proteins. Antibodies that were raised against synthetic peptides whose sequences have been deduced from the ORF were shown to specifically detect the 127-kD tomato RdRP protein. The immunoresponse to the antibodies correlated with the enzymatic activity profile of the RdRP after chromatography on Q-, poly(A)-, and poly(U)-Sepharose, hydroxyapatite, and Sephadex G-200 columns. DNA gel blot analysis revealed a single copy of the RdRP gene in tomato. RdRP homologs from petunia, Arabidopsis, tobacco, and wheat were identified by using polymerase chain reaction. A sequence comparison indicated that sequences homologous to RdRP are also present in the yeast Schizosaccharomyces pombe and in the nematode Caenorhabditis elegans. The previously described induction of RdRP activity upon viroid infection is shown to be correlated with an increased steady state level of the corresponding mRNA. The possible involvement of this heretofore functionally elusive plant RNA polymerase in homology-dependent gene silencing is discussed.     

Diadenosine 5′,5‴-P1,P4-tetraphosphate- (Ap4A-) mediated stimulation of DNA synthesis in extracts from Xenopus laevis oocytes

Diadenosine 5′,5‴-P¹,P⁴-tetraphosphate (Ap₄A) stimulates DNA synthesis in Xenopus laevis oocytes in the presence of activated DNA as template. Besides Ap₄A, other analogues such as Ap₃A, ATP and other derivatives are able to stimulate DNA polymerase activity. The effect of Ap₄A on DNA synthesis is observed with poly(dT) and poly(dT)-poly(dA) as templates, while no effect is found with poly(dA)(dT)_12–18 and poly(dC)(dG)_12–18. In the presence of a poly(dT) template, the oocyte extract is able to utilize Ap₄A as primer and to form a covalent bond between this dinucleotide and the nascent poly(dA) chain. An Ap₄A-binding protein present in the system has been purified and separated from DNA polymerase α-primase after phosphocellulose chromatography. After this separation, Ap₄A is no longer able to stimulate the polymerase activity, or to be utilized as primer by DNA polymerase α-primase.     

Mode of Action of Antibiotic U-20,661

Antibiotic U-20,661 was shown to inhibit predominantly deoxyribonucleic acid (DNA)-directed ribonucleic acid (RNA) synthesis by binding to the double-stranded DNA template. Specific binding to DNA was verified by difference spectroscopy, reversal of the RNA polymerase inhibitory effect by increasing concentrations of DNA template, and by moderately increasing the melting temperature of double-stranded DNA in the presence of the antibiotic. The RNA polymerase reaction primed with synthetic poly dAT was inhibited considerably, but not completely even with high concentrations of antibiotic. Thus, the agent might bind to adenine or thymidine or both bases in the double-stranded DNA helix.