Inhibition of DNA primase by 9-beta-D-arabinofuranosyladenosine triphosphate.

9-beta-D-Arabinofuranosyladenosine triphosphate (araATP) is a potent inhibitor of DNA primase. Primase readily incorporates araATP into primers, and primers containing araAMP are then elongated by DNA polymerase alpha (pol alpha) upon addition of dNTPs. AraATP did not inhibit utilization of primers under conditions where the ability of pol alpha to elongate primers was independent of the dATP concentration. The fraction of primers elongated by pol alpha was reduced by araATP only when elongation was dependent upon the dATP concentration. When the Ki for primase was measured in terms of the inhibition of the synthesis of primers that can be utilized by pol alpha, we obtained Ki = 2.7 microM (37 degrees C) and 2.0 microM (25 degrees C). Inhibition was competitive with ATP. Inhibition of pol alpha activity by araATP was measured under conditions where primase-catalyzed primer synthesis was required for the pol alpha activity. The decreased pol alpha activity was due to primase inhibition, and at constant dATP, araATP inhibition was competitive with ATP and gave Ki = 1.2 microM, similar to the Ki for primase alone. Increasing the dATP concentration had no effect on inhibition. In combination with previously reported in vivo data, we conclude that DNA primase is the primary in vivo target of the arabinofuranosyl nucleotides, not pol alpha.     

Alterations in deoxynucleoside triphosphate metabolism in DNA damaged cells: identification and consequences of poly(ADP-ribose) polymerase dependent and independent processes

Treatment of L1210 cells with increasing concentrations of MNNG produces heterogeneous perturbations of cellular deoxynucleoside triphosphate pools, with the magnitude and direction of the shift depending on the deoxynucleotide and on the concentration and time of exposure of the DNA damaging agent. 5 microM MNNG stimulated an increase in dATP, dCTP and dTTP but dGTP pools remained constant. These increases were not affected by 3-aminobenzamide, indicating that the pool size increases were produced by poly(ADP-ribose) polymerase independent reactions. 30 microM MNNG caused a time dependent decrease in dATP, dGTP, dTTP and dCTP. The dGTP pool was most drastically affected, becoming totally depleted within 3 hours. The fall in all 4 dNTP pools was substantially prevented by 3-aminobenzamide, suggesting that the decrease in dNTPs following DNA damage is mediated by a poly(ADP-ribose) polymerase dependent reaction. Severe depression of dGTP pools consequent to NAD and ATP depletion may provide a metabolic pathway for rapidly stopping DNA synthesis as a consequence of DNA damage and the activation of poly(ADP-ribose) polymerase.     

Effects of triethylene tetraamine on telomerase activity and proliferation in HeLa cells

Telomerase, which is required to maintain telomeres, has attracted considerable attention as a target for anticancer therapy. In this study, we investigated the inhibition of HeLa cell telomerase activity and cell cycle progression by triethylene tetraamine (TETA), using a modified telomeric repeat amplification protocol (TRAP) assay, and flow cytometry. TETA inhibited telomerase activity in HeLa cell extracts, with an IC50 of about 7.8 microM. Coupled with this inhibition, TETA also increased the proportion of cells in the G1 phase of the cell cycle in a dose-dependent manner. These findings demonstrate that TETA is a potent inhibitor of telomerase in micromolar concentrations, and inhibits the proliferation of HeLa cells by arresting them in G1.     

Tankyrase-1 function at telomeres and during mitosis is regulated by Polo-like kinase-1-mediated phosphorylation

Telomere length is critical for chromosome stability that affects cell proliferation and survival. Telomere elongation by telomerase is inhibited by the telomeric protein, TRF1. Tankyrase-1 (TNKS1) poly(ADP-ribosyl)ates TRF1 and releases TRF1 from telomeres, thereby allowing access of telomerase to the telomeres. TNKS1-mediated poly(ADP-ribosyl)ation also appears to be crucial for regulating the mitotic cell cycle. In searching for proteins that interact with polo-like kinase-1 (Plk1) by using complex proteomics, we identified TNKS1 as a novel Plk1-binding protein. Here, we report that Plk1 forms a complex with TNKS1 in vitro and in vivo, and phosphorylates TNKS1. Phosphorylation of TNKS1 by Plk1 appears to increase TNKS1 stability and telomeric poly(ADP-ribose) polymerase (PARP) activity. By contrast, targeted inhibition of Plk1 or mutation of phosphorylation sites decreased the stability and PARP activity of TNKS1, leading to distort mitotic spindle-pole assembly and telomeric ends. Taken together, our results provide evidence of a novel molecular mechanism in which phosphorylation of TNKS1 by Plk1 may help regulate mitotic spindle assembly and promote telomeric chromatin maintenance.     

ATP activation of DNA polymerase III holoenzyme of Escherichia coli. I. ATP-dependent formation of an initiation complex with a primed template

ATP (or dATP) stimulates DNA synthesis by DNA polymerase III holoenzyme (holoenzyme) on the synthetic template-primer poly(dA).oligo(dT)12. Nonhydrolyzable ATP analogs and other natural (deoxy)ribonucleoside triphosphates are inactive. Because the nonhydrolyzable analog 5'-deoxyadenylylimidodiphosphate is efficiently used by holoenzyme for incorporation, the ATP (or dATP) requirement for activation of replication of natural DNA could be determined. Analysis of lag times in DNA synthesis and isolation of intermediates showed that ATP (or dATP) is required in the formation of an initiation complex between holoenzyme and primed DNA template, but not for subsequent DNA synthesis. ATP is bound to holoenzyme in the absence of DNA with a KD value of 0.8 microM; 2 to 3 molecules of ATP per molecule of holoenzyme are bound without apparent cooperativity. Binding of ATP to DNA polymerase III (holoenzyme minus beta subunit) is weak (KD greater than 5 microM) and binding to the beta subunit alone is not observed. However, holoenzyme reconstituted by mixing DNA polymerase III with beta subunit binds ATP as tightly (KD = 0.6 microM) as the original holoenzyme.     

Might telomerase enzyme be a possible target for trans-Pt(II) complexes?

Telomerase is a ribonucleoprotein polymerase that synthesizes telomeric DNA (TTAGGG) repeats. Previously, we have studied the effect on telomerase enzyme of several cis-platinum(II) complexes bearing aromatic amines as bulky carrier groups. All these complexes possess cis-geometry, according to the Cleare and Hoschele's rule. Since recent reports have dealt with the anti-cancer activity of trans-platinum compounds, in this study we have investigated the Farrell's prototypical trans-[Pt(Cl)2(pyridine)2], hereafter called trans-PtPy, in order to understand whether it may possess any anti-telomerase activity. The trans-PtPy has low water solubility and requires dimethyl sulfoxide (DMSO) as co-solvent, thus making the biological tests problematic. The effect of trans-PtPy on MCF-7 cell line concerning log-term telomerase inhibition, telomerase-related gene expression, viability, and apoptosis was evaluated. In a cell-free biochemical assay, trans-PtPy showed significant and dose-dependent inhibition of semi-purified telomerase. The bulk of data indicate that trans-PtPy acts as a non-properly selective anti-proliferative agent, although it shows an initial telomerase inhibitory effect. Telomere length reduction seems not to be the main mechanism causing the observed cell apoptosis. For comparison purpose, results on cis-[Pt(Cl)2(pyridine)2], hereafter cis-PtPy, are reported.     

DNA binding features of human POT1: a nonamer 5'-TAGGGTTAG-3' minimal binding site, sequence specificity, and internal binding to multimeric sites

The human telomeric protein POT1 is known to bind single-stranded telomeric DNA in vitro and to participate in the regulation of telomere maintenance by telomerase in vivo. We examined the in vitro DNA binding features of POT1. We report that deleting the oligosaccharide/oligonucleotide-binding fold of POT1 abrogates its DNA binding activity. The minimal binding site (MBS) for POT1 was found to be the telomeric nonamer 5'-TAGGGTTAG-3', and the optimal substrate is [TTAGGG](n (n > or = 2)). POT1 displays exceptional sequence specificity when binding to MBS, tolerating changes only at position 7 (T7A). Whereas POT1 binding to MBS or [TTAGGG](2) was enhanced by the proximity of a 3' end, POT1 was able to bind to a [TTAGGG](5) array when positioned internally. These data indicate that POT1 has a strong sequence preference for the human telomeric repeat tract and predict that POT1 can bind both the 3' telomeric overhang and the displaced TTAGGG repeats at the base of the t-loop.     

The effect of Cu2+, Zn2+ cations and biogenic amines on the nuclear poly(ADP-ribose) polymerase activity in the rat brain

Study of the effects of Cu2+, Zn2+ cations and polyamines, spermine and spermidine, on the nuclear poly(ADP-ribose)polymerase activity of rat brain was carried out. It was shown that low concentrations of Cu2+ stimulate the activity of purified poly(ADP-ribose)polymerase. The poly(ADP-ribose)polymerase activity was increased 1.4-fold at 5 microM Cu2+. A further increase of Cu2+ concentration inhibited the enzymatic activity; at 50 microM Cu2+ the polymerase activity appeared to be fully inhibited. It was shown that Zn2+ inhibited only the poly(ADP-ribose)polymerase activity. Zn2+ at a concentration of 125 microM fully inhibited the enzymatic activity. Spermine and spermidine stimulated the poly(ADP-ribose)polymerase activity of brain nuclei of newborn and old rats.     

Purification and characterization of porcine liver DNA polymerase gamma: utilization of dUTP and dTTP during in vitro DNA synthesis.

Porcine liver DNA polymerase gamma has been demonstrated to preferentially incorporate dTMP over dUMP during in vitro DNA synthesis. When polymerase activity was measured in standard reactions containing saturating levels of either dTTP or dUTP, the polymerization rate was slightly faster in the reaction containing dTTP. However, under conditions where both dTTP and dUTP competed, at an equal molar concentration, approximately 3-times more thymine residues were incorporated than uracil residues into DNA. Similarly, preferential incorporation of dTMP was observed on several substrates including poly (dA).oligo p(dT), poly (rA).oligo p(dT) and poly (dA-dT). The discrimination against dUMP incorporation was even more apparent with reduced levels of dUTP. These observations were consistent with the finding that the Km for DNA polymerase gamma was about 3-fold lower for dTTP (0.4 microM) than for dUTP (1.1 microM). On the other hand, the Vmax for these two reactions was very similar. Discrimination against dUMP incorporation was also observed during inhibition of polymerase gamma by dideoxyribonucleoside triphosphates. Dideoxythymidine triphosphate preferentially inhibited dUMP incorporation compared to that of dTMP, whereas ddATP, ddCTP and ddGTP inhibited both reactions equally.     

Synthetic nucleosides and nucleotides. 43. Inhibition of vertebrate telomerases by carbocyclic oxetanocin g (C.OXT-G) triphosphate analogues and influence of C.OXT-G treatment on telomere length in human HL60 cells

Telomerase, responsible for telomere synthesis, is expressed in approximately 90% of human tumor cells but seldom in normal somatic cells. In this study, inhibition by carbocyclic oxetanocin G triphosphate (C. OXT-GTP) and its analogues was investigated in order to clarify the susceptibility of telomerase to various nucleotide analogues. C. OXT-GTP competitively inhibited telomerase activity with respect to dGTP However, C. OXT-GTP had a potent inhibitory effect on DNA polymerase alpha. It was examined whether the nucleoside (C. OXT-G) was able to alter telomere length in cultured human HL60 cells. Contrary to expectation, long-term treatment with 10 microM C. OXT-G was found to cause telomere lengthening.     

Interaction of an acridine dimer with DNA quadruplex structures.

The reactivation of telomerase activity in most cancer cells supports the concept that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. The telomeric G-rich single-stranded DNA can adopt an intramolecular G-quadruplex structure in vitro, which has been shown to inhibit telomerase activity. The C-rich sequence can also adopt a quadruplex (intercalated) structure (i-DNA). Two acridine derivatives were shown to increase the melting temperature of the G- quadruplex and the C-quadruplex at 1 microM dye concentration. The increase in Tm value of the G-quadruplex was associated with telomerase inhibition in vitro. The most active compound, "BisA", showed an IC(50) value of 0.75 microM in a standard TRAP assay.     

Effects of nucleotide analogues on Euplotes aediculatus telomerase processivity: evidence for product-assisted translocation.

Telomerase is a unique ribonucleoprotein that reverse transcribes a defined region of its RNA subunit onto the ends of eukaryotic chromosomes. The product of telomerase, telomeric DNA, is typically a G-rich repeated sequence, (TTTTGGGG)(n) in the ciliate Euplotes aediculatus and (TTAGGG)(n) in humans. Telomerase can extend oligonucleotide primers in vitro in a processive fashion. We used dNTP analogues to study the structure-activity relationship between substrate nucleotides and processivity of telomerase from E. aediculatus. Several analogues, including 2'-deoxyuridine triphosphate (dUTP), 2'-deoxyinosine triphosphate (dITP), and 7-deaza-2'-deoxyguanosine triphosphate (7-deaza-dGTP), were good substrates for telomerase with K(m) and V(max) values near those of the natural substrates, dTTP and dGTP. However, telomerase processivity was affected with these substrates, decreasing in the order dUTP > 7-deaza-dGTP > dITP. Telomerase did not completely reverse transcribe the template when dITP was the substrate, and it efficiently extended a primer by the addition of two repeats when 7-deaza-dGTP and dUTP were utilized. When the same nucleotide analogues were incorporated into the primers, no effects were observed except in the case of a 3'-terminal deoxyinosine. The data support a model that includes the formation of an intramolecular secondary structure within the product DNA to facilitate translocation. The most likely structure is a G-G hairpin.     

Rapid regulation of telomere length is mediated by poly(ADP-ribose) polymerase-1

Shelterin/telosome is a multi-protein complex at mammalian telomeres, anchored to the double-stranded region by the telomeric-repeat binding factors-1 and -2. In vitro modification of these proteins by poly(ADP-ribosyl)ation through poly(ADP-ribose) polymerases-5 (tankyrases) and -1/-2, respectively, impairs binding. Thereafter, at least telomeric-repeat binding factor-1 is degraded by the proteasome. We show that pharmacological inhibition of poly(ADP-ribose) polymerase activity in cells from two different species leads to rapid decrease in median telomere length and stabilization at a lower setting. Specific knockdown of poly(ADP-ribose) polymerase-1 by RNA interference had the same effect. The length of the single-stranded telomeric overhang as well as telomerase activity were not affected. Release of inhibition led to a fast re-gain in telomere length to control levels in cells expressing active telomerase. We conclude that poly(ADP-ribose) polymerase-1 activity and probably its interplay with telomeric-repeat binding factor-2 is an important determinant in telomere regulation. Our findings reinforce the link between poly(ADP-ribosyl)ation and aging/longevity and also impact on the use of poly(ADP-ribose) polymerase inhibitors in tumor therapy.     

Telomerase activity and telomere detection during early bovine development

The ends of mammalian chromosomes are composed of repeated DNA sequences of (TTAGGG)(n) known as telomeres. Telomerase is a ribonucleoprotein that synthesizes telomeric DNA to replenish the 50-200 bp lost during cell replication. Cellular aging and senescence are associated with a lack of telomerase activity and a critical shortening of the telomere. The objectives of this study were to confirm the presence of TTAGGG repeats on the chromosomes of bovine embryos using in situ hybridization and assess the relative amounts of telomerase activity using a telomeric repeat amplification protocol (TRAP) during oocyte maturation and early embryo development. Applying a telomere DNA probe to the chromosomes of blastocysts and adult fibroblasts, telomeres were identified on the terminal ends of the p and q arms of chromosomes in all cells examined. Immature oocytes, matured oocytes, zygotes, 2- to 5-cell embryos, 6- to 8-cell embryos, morulae, and blastocysts were lysed in NP-40 lysis buffer and telomerase activity was assayed using the TRAP assay. Telomerase activity was detected in all developmental stages examined. Relative telomerase activity (based on telomerase internal standards and positive controls) appeared to decrease during oocyte maturation and subsequent development to the 8-cell stage but significantly increased (P < 0.05) by approximately 40-fold at the morula and blastocyst stages. It was concluded that the telomeres of bovine chromosomes contain TTAGGG repeats and that telomerase activity is up-regulated in morulae and blastocysts.     

Analysis of inhibitors of bacteriophage T4 DNA polymerase.

Bacteriophage T4 DNA polymerase was inhibited by butylphenyl nucleotides, aphidicolin and pyrophosphate analogs, but with lower sensitivities than other members of the B family DNA polymerases. The nucleotides N2-(p-n-butylphenyl)dGTP (BuPdGTP) and 2-(p-n-butylanilino)dATP (BuAdATP) inhibited T4 DNA polymerase with competitive Ki values of 0.82 and 0.54 microM with respect to dGTP and dATP, respectively. The same compounds were more potent inhibitors in truncated assays lacking the competitor dNTP, displaying apparent Ki values of 0.001 and 0.0016 microM, respectively. BuPdGTP was a substrate for T4 DNA polymerase, and the resulting 3'-BuPdG-primer:template was bound strongly by the enzyme. Each of the non-substrate derivatives, BuPdGDP and BuPdGMPCH2PP, inhibited T4 DNA polymerase with similar potencies in both the truncated and variable competitor assays. These results indicate that BuPdGTP inhibits T4 DNA polymerase by distinct mechanisms depending upon the assay conditions. Reversible competitive inhibition predominates in the presence of dGTP, and incorporation in the absence of dGTP leads to potent inhibition by the modified primer:template. The implications of these findings for the use of these inhibitors in the study of B family DNA polymerases is discussed.