Inhibition of DNA polymerase α by gossypol

Our earlier studies have shown that gossypol is a specific inhibitor of DNA synthesis in cultured cells at low doses. In an attempt to determine the mechanism for the inhibition of DNA synthesis by gossypol we observed that gossypol does not interact with DNA per se but may affect some of the enzymes involved in DNA replication. These studies indicated that gossypol inhibits both in vivo and in vitro the activity of DNA polymerase α (EC 2.7.7.7), a major enzyme involved in DNA replication, in a time- and dose-dependent manner. Kinetic analysis revealed that gossypol acts as a noncompetitive inhibitor of DNA polymerase α with respect to all four deoxynucleotide triphosphates and to the activated DNA template. Inhibition of DNA polymerase α does not appear to be due to either metal chelation or reduction of sulfhydryl groups on the enzyme. Gossypol also inhibited HeLa DNA polymerase β in a dose-dependent manner, but had no effect on DNA polymerase γ. These results suggest that inhibition of DNA polymerase α may account in part for the inhibition of DNA synthesis and the S-phase block caused by gossypol. The data also raise the possibility that gossypol may interfere with DNA repair processes as well.     

Photoaffinity labeling of ribulose-bisphosphate carboxylase/oxygenase with 8-azidoadenosine 5′-triphosphate

Photoaffinity labeling with [³²P] 8-azidoadenosine 5-triphosphate (8-N₃ATP) was used to identify putative binding sites on tobacco (Nicotiana tabacum L. and N. rustica L.) leaf ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase, EC 4.1.1.39). Incorporation of ³²P was observed in polypeptides corresponding to both RuBPCase subunits when desalted leaf and chloroplast extracts, and purified RuBPCase were irradiated with ultraviolet light in the presence of [³²P] 8-N₃ATP. ³²P-labeling was dependent upon ultraviolet irradiation and occurred with [³²P] 8-N₃ATP labeled in the -position, indicating covalent incorporation of the photoprobe. Both [³²P] 8-N₃ATP and [³²P] 8-N₃GTP were incorporated to a similar extent into the 53-kilodalton (kDa) large subunit (LSu), but incorporation of [³²P] 8-N₃GTP into the 14-kDa small subunit (SSu) of RuBPCase was <5% of that measured with [³²P] 8-N₃ATP. Distinct binding sites for 8-N₃ATP on the two subunits were indicated by different apparent K _D values, 3 and 18 M for the SSu and LSu, respectively, and differences in the response of photoaffinity labeling to Mg²⁺, anions and enzyme activation. Active-site-directed compounds, including the non-gaseous substrate ribulose 1,5-bisphosphate, the reaction intermediate analog 2-carboxyarabinitol-1,5-bisphosphate and several phosphorylated effectors afforded protection to the LSu site against photoincorporation but provided almost no protection to the SSu. These results indicate that 8-N₃ATP binds to the active-site region of the LSu and a distinct site on the SSu of RuBPCase. Experiments conducted with intact pea (Pisum sativum L.) and tobacco chloroplasts showed that the SSu was not photolabeled with [³²P] 8-N₃ATP in organello or in undesalted chloroplast lysates but was photolabeled when lysates were ultrafiltered or desalted. These results indicate that 8-N₃ATP binds to a site on the SSu that has physiological significance.Abbreviations kDa kilodalton - LSu large subunit - 8-N₃ATP 8-azidoadenosine 5-triphosphate - RuBP ribulose-1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase - SSu small subunit Kentucky Agricultural Experiment Station Journal Article No. 89-3-150The authors acknowledge the technical assistance of J.C. Anderson. This work was supported in part by National Institute of Health grant GM 35766 to B.E.H.     

Inactivation of DNA polymerases by adenosine 2′,3′-riboepoxide 5′-triphosphate allows estimation of the primers affinity

Template-primer dependent inactivation of human DNA polymerase and Klenow fragment of E. coli DNA polymerase I by adenosine 2,3-riboepoxide 5-triphosphate was used for quantitative analysis of the K_d values for oligonucleotide primers of different length. The K_d values are smaller by a factor of 2.5 than the K_m values for the same primers determined in the reaction of DNA polymerization in the case of DNA polymerase . The K_d and K_m values are nearly the same for Klenow fragment. Such approach to the determination of K_m/K_d ratio can likely be used for detailed quantitative analysis of DNA polymerases.Abbreviations epATP adenosine 2,3-riboepoxide 5-triphosphate - KF Klenow fragment of E. coli DNA polymerase I - Pol I E. coli DNA polymerase I - Pol human placenta DNA polymerase      

Inhibition of calf thymus DNA polymerase α and of normal and cancer cell growth by butylanilinouracil and butylphenylguanine

6-(p-n-Butylanilino)uracil and N²-(p-butylphenyl)guanine inhibited the activity of DNA polymerase α from calf thymus but had no effect on other eukaryotic polymerases (DNA polymerases β and γ) or Escherichia coli DNA polymerase I. Inhibition was competitive with deoxyguanosine 5′-triphosphate and did not occur in the reaction of DNA polymerase α with a template that did not contain cytosine residues. The results support a mechanism which involves hydrogen bonding of inhibitors with cytosines in the DNA template and binding with an inhibitor specific site on the enzyme. A screen of inhibitor effects on normal and cancer cell growth in culture showed that cells were not uniformly sensitive to these compounds, a mouse lymphoma line being least sensitive and a human lung cancer line being most sensitive. It is suggested that these inhibitors may be useful to probe possible structural differences among DNA polymerases α.     

2′-Deoxy-1-methylpseudocytidine,a stable analog of 2′-deoxy-5-methylisocytidine

2′-Deoxy-5-methylisocytidine is widely used in assays to personalize the care of patients infected with HIV, hepatitis C, and other infectious agents. However, oligonucleotides that incorporate 2′-deoxy-5-methylisocytidine are expensive, because of its intrinsic chemical instability. We report here a C-glycoside analog that is more stable and, in oligonucleotides, pairs with 2′-deoxyisoguanosine, contributing to duplex stability about as much as a standard 2′-deoxycytidine and 2′-deoxyguanosine pair.     

Regulation of yeast DNA polymerase δ-mediated strand displacement synthesis by 5′-flaps

The strand displacement activity of DNA polymerase δ is strongly stimulated by its interaction with proliferating cell nuclear antigen (PCNA). However, inactivation of the 3′–5′ exonuclease activity is sufficient to allow the polymerase to carry out strand displacement even in the absence of PCNA. We have examined in vitro the basic biochemical properties that allow Pol δ-exo⁻ to carry out strand displacement synthesis and discovered that it is regulated by the 5′-flaps in the DNA strand to be displaced. Under conditions where Pol δ carries out strand displacement synthesis, the presence of long 5′-flaps or addition in trans of ssDNA suppress this activity. This suggests the presence of a secondary DNA binding site on the enzyme that is responsible for modulation of strand displacement activity. The inhibitory effect of a long 5′-flap can be suppressed by its interaction with single-stranded DNA binding proteins. However, this relief of flap-inhibition does not simply originate from binding of Replication Protein A to the flap and sequestering it. Interaction of Pol δ with PCNA eliminates flap-mediated inhibition of strand displacement synthesis by masking the secondary DNA site on the polymerase. These data suggest that in addition to enhancing the processivity of the polymerase PCNA is an allosteric modulator of other Pol δ activities.     

Inhibition of shoot induction by 5-azacytidine and 5-aza-2′-deoxycytidine in Petunia involves DNA hypomethylation

Shoot bud regeneration from Petunia leaf disks was inhibited when they were cultured with the demethylating agents, 5-azacytidine (AzaC) and 5-aza-2′-deoxycytidine (AzadC), in shoot induction (SI) medium. Explants induced shoot primordia if they were transferred after 1 week from the medium containing the drugs to medium without drugs. The fresh weight of leaf disks cultured on SI medium for 2 weeks in the presence of the drugs was 60–80% lower when compared to control shoot-forming cultures. Internode length was reduced when shoots were transferred to phytohormone-free Murashige and Skoog medium containing the drugs. However, no other morphological abnormalities were seen in these shoots, even at 20 μm AzaC or 5 μm AzadC. Coupled restriction enzyme digestion (with HpaII and MspI) and random amplification of genomic DNA was performed to detect the level of methylation of CCGG sites in the DNA of the explants exposed to AzaC and AzadC. Over 15 amplified bands were detectable in the control. Five of these bands were absent in the amplified products when digested DNA from the drug-treated explants was used as the template, showing that hypomethylation of DNA had occurred. This suggests that inhibition of shoot bud formation in the presence of the drugs AzaC and AzadC may be due to the altered methylation status. Received: 7 January 1997 / Revision received: 17 February 1997 / Accepted: 1 March 1997     

Modulation of diphthamide synthesis by 5′-deoxy-5′-methylthioadenosine in murine lymphoma cells

The histidine derivative diphthamide occurs uniquely in eukaryotic elongation factor 2 (EF-2), and is the specific target for the diphtheria toxin mono(ADP-ribosyl)transferase. The first step in diphthamide biosynthesis may involve the transfer of an aminocarboxypropyl moiety from S-adenosylmethionine to the imidazole ring of histidine in EF-2, to yield 2-(3-carboxy-3-aminopropyl)histidine and 5′-deoxy-5′-methylthioadenosine (MeSAdo). As the possible nucleoside product of the initial reaction in the diphthamide biosynthetic pathway, MeSAdo could be an inhibitor of diphthamide formation. In the present experiments, we have analyzed the effects of MeSAdo on diphthamide synthesis in a MeSAdo phosphorylase-deficient mutant murine lymphoma cell line (R1.1, clone H3). As measured by susceptibility to diphtheria toxin-induced ADP-ribosylation, MeSAdo inhibited the formation of diphthamide in EF-2. The inhibition was not due to a nonspecific effect on protein synthesis. Indeed, exogenous MeSAdo substantially protected the lymphoma cells from the lethal effects of diphtheria toxin. These results suggest that MeSAdo can specifically modulate the biosynthesis of diphthamide in EF-2 in murine malignant lymphoma cells.     

Carborane-linked 2′-deoxyuridine 5′-O-triphosphate as building block for polymerase synthesis of carborane-modified DNA

5-[(p-Carborane-2-yl)ethynyl]-2′-deoxyuridine 5′-O-triphosphate was synthesized and used as a good substrate in enzymatic construction of carborane-modified DNA or oligonucleotides containing up to 21 carborane moieties in primer extension reactions by DNA polymerases.     

Analysis of DNA Sequencing Reaction Products Made with 7-Halo-7-deaza-2′-deoxyguanosine-5′-triphosphate

Abstract

7-Chloro- and 7- Iodo-7-Deaza-2′-Deoxyguanosine-5′-Triphosphates were employed as substrates replacing dGTP, dITP, or 7-Deaza-2′-Deoxyguanosine-5′-Triphosphate in sequencing reactions with Thermo Sequenase^TM. Analysis of the reaction products by denaturing gel electrophoresis indicates DNA containing these halogenated analogs can form strong secondary structures.     

Automated parallel synthesis of 5′-triphosphate oligonucleotides and preparation of chemically modified 5′-triphosphate small interfering RNA

A fully automated chemical method for the parallel and high-throughput solid-phase synthesis of 5′-triphosphate and 5′-diphosphate oligonucleotides is described. The desired full-length oligonucleotides were first constructed using standard automated DNA/RNA solid-phase synthesis procedures. Then, on the same column and instrument, efficient implementation of an uninterrupted sequential cycle afforded the corresponding unmodified or chemically modified 5′-triphosphates and 5′-diphosphates. The method was readily translated into a scalable and high-throughput synthesis protocol compatible with the current DNA/RNA synthesizers yielding a large variety of unique 5′-polyphosphorylated oligonucleotides. Using this approach, we accomplished the synthesis of chemically modified 5′-triphosphate oligonucleotides that were annealed to form small-interfering RNAs (ppp-siRNAs), a potentially interesting class of novel RNAi therapeutic tools. The attachment of the 5′-triphosphate group to the passenger strand of a siRNA construct did not induce a significant improvement in the in vitro RNAi-mediated gene silencing activity nor a strong specific in vitro RIG-I activation. The reported method will enable the screening of many chemically modified ppp-siRNAs, resulting in a novel bi-functional RNAi therapeutic platform.     

Reinvestigation of 4-Thiothymidine-5′-triphosphate Synthesis

Abstract

The 4-Thiothymidine-5′-triphosphate 1 (S⁴TTP) was known to be a substrate for polymerase, however a commercial sample of this compound failed to be incorporated into DNA. Mass spectrometry combined to alkaline phosphatase digestion and ³¹P-NMR showed that this sample was in fact 5′-chloro-5′-deoxy-4-thiothymidine-3′-triphosphate 2 . The desired S⁴TTP was synthesized by two alternate routes, was fully characterized and was shown to be incorporated in a DNA polymerase assay.     

Biochemical properties of the stimulatory activity of DNA polymerase α by the hyper-phosphorylated retinoblastoma protein

Previously, my colleagues and I have reported that the immunopurified hyper-phosphorylated retinoblastoma protein (ppRb) stimulates the activity of DNA polymerase α. I describe here the biochemical characteristics of this stimulatory activity. DNA polymerase α-stimulatory activity of ppRb was most remarkable when using activated DNA as a template-primer, rather than using poly(dT)-(rA)₁₀, poly(dA)-(dT)_12–18, and so on. Kinetic analysis showed that there was no significant difference in K_m value for deoxyribonucleotides of DNA polymerase α in the presence of ppRb. Adding ppRb resulted in the overcoming pause site on the template, but did not affect the rate of misincorporation of incorrect deoxyribonucleotides. By adding ppRb, the optimal concentration of template-primer was shifted to a higher region, but not using M13 singly primed DNA. The ppRb seemed to assist the process that DNA polymerase α changed its conformation resulting in appropriate enzyme activity. These results suggest that ppRb affects both template-primer and DNA polymerase α and makes appropriate circumstances for the enzyme reaction.     

Synthesis and Properties of Oligonucleotide Chimeras Containing 5′-Amino-2′-deoxy-2′-fluoroarabinonucleosides

Abstract

Oligonucleotide analogues comprised of 2′-deoxy-2′-fluoro-β-D-arabinose units joined via P3′-N5′ phosphoramidate linkages (2′F-ANA^5′N) were prepared for the first time. Among the compounds prepared were a series of 2′OMe-RNA-[GAP]-2′OMe-RNA ‘chimeras’, whereby the “GAP” consisted of DNA, DNA^5′N, 2′F-ANA or 2′F-ANA^5′N segments. The chimeras with the 2′F-ANA and DNA gaps exhibited the highest affinity towards a complementary RNA target, followed by the 5′-amino derivatives, i.e., 2′F-ANA > DNA > 2′F-ANA^5′N > DNA^5′N. Importantly, hybrids between these chimeras and target RNA were all substrates of both human RNase HII and E.coli RNase HI. In terms of efficiency of the chimera in recruiting the bacterial enzyme, the following order was observed: gap DNA > 2′F-ANA > 2′F-ANA^5′N > DNA^5′N. The corresponding relative rates observed with the human enzyme were: gap DNA > 2′F-ANA^5′N > 2′F-ANA > DNA^5′N.