Calf Thymus Terminal Deoxynucleotidyl Transferase Research Articles

New Triphosphate Conjugates Bearing Reporter Groups: Labeling of DNA Fragments for Microarray Analysis

A simple and convenient method for incorporation of fluorescent or ligand groups into 3'-termini of DNA fragments is proposed. A set of triphosphoric acid monoesters bearing fluorescent groups or biotin attached to the triphosphate fragment through linkers of different lengths and structures was synthesized. All the compounds were substrates for calf thymus terminal deoxynucleotidyltransferase and were used for incorporation of marker groups into 3'-termini of DNA fragments. The compounds were successfully applied for DNA labeling during post-PCR target preparation for microarray analysis.

New Nonnucleoside Substrates for Terminal Deoxynucleotidyl Transferase: Synthesis and Dependence of Substrate Properties on Structure

N-(9-Fluorenylmethoxycarbonyl)-omega-aminoalkyl, N-(9-fluorenylmethoxycarbonyl)-8-amino-3,6-dioxaoctyl, and N-(9-fluorenylmethoxycarbonyl)-6-aminohexanoyl]-2-aminoethyl triphosphates were synthesized. All of them were shown to be the substrates of the calf thymus terminal deoxynucleotidyl transferase. Their substrate properties depend on the length and structure of linker between the 9-fluorenylmethoxycarbonyl and triphosphate moieties.

DNA Elongation by the Human DNA Polymerase λ Polymerase and Terminal Transferase Activities Are Differentially Coordinated by Proliferating Cell Nuclear Antigen and Replication Protein A

DNA polymerase lambda contains template-dependent (DNA polymerase) and template-independent (terminal transferase) activities. In this study we enzymologically characterized the terminal transferase activity of polymerase lambda (pol lambda-tdt). Pol lambda-tdt activity was strongly influenced by the nature of the 3'-terminal sequence of the DNA substrate, and it required a single-stranded (ss) DNA 3'-overhang of about 9-12 nucleotides for optimal activity. The strong preference observed for pyrimidine versus purine nucleotide incorporation was found to be due, at least partially, to a steric block imposed by the residue Tyr-505 in the active site of pol lambda. Pol lambda-tdt was found to be able to elongate a 3'-ssDNA end by two alternative mechanisms: first, a template-independent one resulting in addition of 1 or 2 nucleotides, and second, a template-dependent one where a homopolymeric tract as short as 3 nucleotides at the 3'-end could be used as a template to direct DNA polymerization by a looping back mechanism. Furthermore repetitive cycles of DNA synthesis resulted in the expansion of such a short homopolymeric terminal sequence. Most importantly we found that the proliferating cell nuclear antigen was able to selectively block the looping back mechanism while stimulating the single terminal nucleotide addition. Finally replication protein A completely suppressed the transferase activity of pol lambda while stimulating the polymerase activity, suggesting that proliferating cell nuclear antigen and replication protein A can coordinate the polymerase and the terminal transferase activities of pol lambda.

A novel DNA topoisomerase inhibitor: dehydroebriconic acid, one of the lanostane-type triterpene acids from Poria cocos.

Traditional Chinese medicinal plants are a treasure house for screening novel inhibitors of DNA polymerases and DNA topoisomerases from mammals; in the present study, nine lanostane-type triterpene acids were found in sclerotium of Poria cocos. Among the nine compounds, only dehydroebriconic acid could potently inhibit DNA topoisomerase II (topo II) activity (IC(50) = 4.6 microM), while the compound moderately inhibited the activities of DNA polymerases alpha, beta, gamma, delta, epsilon, eta, iota, kappa and lambda only from mammals, to similar extents. Another compound, dehydrotrametenonic acid, also showed moderate inhibitory effects against topo II (IC(50) = 37.5 microM) and weak effects against all the polymerases tested. Both compounds showed no inhibitory effect against topo I, higher plant (cauliflower) DNA polymerase I (alpha-like polymerase) or II (beta-like polymerase), calf thymus terminal deoxynucleotidyl transferase, human immunodeficiency virus type-1 reverse transcriptase, prokaryotic DNA polymerases such as the Klenow fragment of E. coli DNA polymerase I, Taq DNA polymerase and T4 DNA polymerase, or DNA metabolic enzymes such as T 7 RNA polymerase, T4 polynucleotide kinase and bovine deoxyribonuclease I. These findings suggest that dehydroebriconic acid and dehydrotrametenonic acid should be designated as topo II-preferential inhibitors, although they also moderately inhibited all the mammalian DNA polymerases tested. Both dehydrotrametenonic acid and dehydroebriconic acid could prevent the growth of human gastric cancer cells, and their LD(50) values were 63.6 and 38.4 microM, respectively. The cells were halted at the G1 phase in the cell cycle. The relation between the structure of triterpene acids and their inhibitory activities is discussed.

3′‐End‐labeling procedure for phosphorothioate oligonucleotides and oligonucleotide‐conjugates

AbstractOligonucleotides offer an enormous potential for therapeutical and diagnostic applications. Unfortunately the cellular uptake is a fundamental problem for the application of antisense oligonucleotides. Oligonucleotides are large charged molecules which are unable to penetrate the cellular membrane by diffusion. The resulting low cellular uptake can be circumvented by employing derivatized oligonucleotides. For this purpose we have recently developed oligonucleotide derivatives which are conjugated to octreotate, a cyclic octapeptide (1). Upon binding to somatostatin receptors (known to be overexpressed in various tumors), these conjugates can be internalized by receptor mediated endocytosis. Therefore the new conjugates consist of the oligonucleotide, which is linked via the 5′‐end to the N‐terminus of octreotate. The oligonucleotide sequence is complementary to the sequence of the protooncogene bcl‐2, which is known to be overexpressed in lymphoma. To monitor the fate of modified oligonucleotides in vivo as well as to enable the use of modified oligonucleotides for the scintigraphic imaging, labeling procedures had to be developed. This procedure also had to be suitable for the labeling of phosphorothioate‐modified oligonucleotides. Due to pharmacological advantages as compared to unmodified phosphodiester oligonucleotides, these synthetic oligonucleotides are utilized for in vivo applications. Using [γ‐32P]‐ATP and T4 polynucleotide kinase, oligonucleotides can be conveniently labeled at the 5′‐end. In order to enable the labeling of 5′‐conjugated oligonucleotides at the 3′‐end, the labeling with calf thymus terminal deoxynucleotidyl transferase (terminal transferase) and dideoxyadenosine‐5′‐[α‐32P]triphosphate ([α‐32P]‐ddATP) was investigated.

Galactosyldiacylglycerol, a mammalian DNA polymerase alpha-specific inhibitor from a sea alga, Petalonia bingbamiae.

The glycolipid galactosyldiacylglycerol (GDG), containing C16:0 and C18:1 fatty acids, was isolated from the sea alga Petalonia bingbamiae as a potent inhibitor of the activities of mammalian DNA polymerase alpha (pol. alpha). GDG, however, had no effect on pol. alpha from a fish or a higher plant. The inhibition of pol. alpha by GDG was dose-dependent with an IC50 value of 54 microM. The compound did not influence the activities of other replicative DNA polymerases such as mammalian pol. delta, or repair-related enzymes such as mammalian pol. beta. GDG also did not influence the activities of prokaryotic DNA polymerases such as the Klenow fragment of DNA polymerase I, T4 DNA polymerase, Taq DNA polymerase, DNA polymerases from the higher plant, cauliflower, or DNA metabolic enzymes such as calf thymus terminal deoxynucleotidyl transferase, human immunodeficiency virus type 1 reverse transcriptase and deoxyribonuclease 1. Kinetic analysis of the compound showed that pol. alpha was non-competitively inhibited with respect to both the DNA template and the nucleotide substrate. In this study, we demonstrated the structure-function relationship in the selective inhibition of pol. alpha by the glycolipid group.

Pyrophosphoryl Derivatives of 1-(2-Deoxy-3-O-Phosphonomethyl-β-and-α-D-Erythro-Pentofuranosyl)Thymine: Synthesis and Substrate Properties Towards Some DNA Polymerases

The synthesis of 1-(2-deoxy-3-O-phosphonomethyl-β-D-erythro-pentofuranosyl)thymine (17) and its α-anomer 18 is described. Attempts to prepare 1-[2-deoxy-3-O-(pyrophosphoryl)phosphonomethyl-β-D-erythro- Pentofuranosyl]thymine (19) by an activation of the respective phosphonate 17 with 1,1′-carbonyldiimidazole (Im2CO) resulted in the quantitative formation of the corresponding pyrophosphonate derivative 21 (Scheme 2). Activation of inorganic pyrophosphate with Im2CO followed by the condensation with the phosphonates 17 and 18 afforded the desired analogues of nucleoside triphosphate 19 (35%) and its α-anomer 20 (27%) along with the respective pyrophosphonate derivatives 21 (37%) and 24 (38%) (Scheme 3). It was found that compounds 19 and 20 display (i) no substrate properties toward calf thymus terminal deoxynucleotidyl transferase (TDT) and AMV reverse transcriptase, and (ii) moderate substrate activity with E. coli DNA polymerase I (Klenow fragment).

Novel anti-inflammatory compounds from Myrsine seguinii, terpeno-benzoic acids, are inhibitors of mammalian DNA polymerases

Novel anti-inflammatory compounds, terpeno-benzoic acids, were found from the plant, Myrsine seguinii. The strongest of these anti-inflammatory agents, 3-geranyl-4-hydroxy-5-(3′-methyl-2′-butenyl) benzoic acid (compound 1), showed an inhibitory effect against enzymes involved in replication, such as calf DNA polymerase α (pol. α), rat DNA polymerase β (pol. β) and one of the β family polymerases, calf thymus terminal deoxynucleotidyl transferase (TdT). The minimum inhibitory concentration (MIC) and IC 50 values were 82 and 22 μM for pol. α, 86 and 11 μM for pol. β, 140 and 46 μM for TdT, respectively. However, compound 1 did not influence the activities of plant DNA polymerases, human immunodeficiency virus type-1 reverse transcriptase, any of the prokaryotic DNA polymerases or DNA and RNA metabolic enzymes tested. Dose-dependent relationships were observed between the anti-inflammatory activities and the DNA polymerase-inhibitory activities of the four derivatives. The carboxylic acid moiety in the benzoic acid of the compounds appeared to be related to the inhibitory effects. The mode of action of the terpeno-benzoic acids against the polymerases and their relationships to the anti-inflammatory activity are discussed.

Terminal deoxynucleotidyl transferase catalyzes the reaction of DNA phosphorylation.

The reaction of phosphorylation and phosphonylation of an oligodeoxynucleotide 3'-terminal hydroxyl (oligodeoxynucleotidyl kinase activity) catalyzed by calf thymus terminal deoxynucleotidyl transferase (TDT) was found. Triphosphates modified at Palpha-, Palpha,gamma- or Palpha,beta,gamma-residues served as low-molecular weight substrates. The reaction was TDT specific; human DNA polymerasesalphaandbeta, as well as AMV reverse transcriptase did not catalyze it. The donor activity of modified triphosphates or triphosphonates depended on their structure and was increased with an increase in their hydrophobicity. The substrate activity of some modified triphosphates was up to one order of magnitude higher than that of ddTTP.

Stereochemical control of DNA biosynthesis.

Stereochemical control of DNA biosynthesis was studied using several DNA-synthesizing complexes containing, in each case, a single substitution of a 2'-deoxy-D-nucleotide residue by an enantiomeric L-nucleotide residue in a DNA chain (either in the primer or in the template) as well as 2'-deoxy-L-ribonucleoside 5'-triphosphates (L-dNTPs) as substrates. Three template-dependent DNA polymerases were tested, Escherichia coli DNA polymerase I Klenow fragment, Thermus aquaticus DNA polymerase and avian myeloblastosis virus reverse transcriptase, as well as template-independent calf-thymus terminal deoxynucleotidyl transferase. Very stringent control of stereoselectivity was demonstrated for template-dependent DNA polymerases, whereas terminal deoxynucleotidyl transferase was less selective. DNA polymerase I and reverse transcriptase catalyzed formation of dinucleoside 5',5'-tetraphosphates when L-dTTP was used as substrate. Comparison between models of template-primer complexes, modified or not by a single L-nucleotide residue, revealed striking differences in their geometry.

4-hydroxy-17-methylincisterol, an inhibitor of DNA polymerase-α activity and the growth of human cancer cells in vitro

An ergosterol derivative, 4-hydroxy-17-methylincisterol (HMI), was found to be an inhibitor of mammalian DNA polymerases in vitro. HMI inhibited the activity of calf thymus DNA polymerase α (pol. α). Among the polymerases tested, pol. α was the most sensitive to inhibition by HMI, and the inhibition was concentration dependent. The inhibitory effect of HMI on pol. α was almost the same as that shown by aphidicolin, a well-known potent pol. α inhibitor. HMI had relatively less effect on rat DNA pol. β, human immunodeficiency virus type 1 reverse transcriptase (HIV-RT), and calf thymus terminal deoxynucleotidyl transferase (TdT) in vitro, and did not influence the activities of prokaryotic DNA polymerases such as Klenow Fragment of DNA polymerase I, or the DNA-metabolic enzyme DNase I. HMI was found to be able to prevent the growth of human cancer cell lines originating from patients with leukemia or various solid tumors; its ic 50 values ranged from 7.5 to 12 μM. We also synthesized other ergosterol derivatives and tested them, and found that two compounds, 17-methylincisterol and 4-acetyl-17-methylincisterol, have similar inhibitory effects.

Studies on inhibitors of mammalian DNA polymerase α and β: Sulfolipids from a pteridophyte, Athyrium niponicum

Three sulfolipid compounds, 1, 2 and 3, have been isolated from a higher plant, a pteridophyte, Athyrium niponicum, as potent inhibitors of the activities of calf DNA polymerase α and rat DNA polymerase β. The inhibition by the sulfolipids was concentration dependent, and almost complete inhibition of DNA polymerase α and DNA polymerase β was achieved at 6 and 8 μg/mL, respectively. The compounds did not influence the activities of calf thymus terminal deoxynucleotidyl transferase, prokaryotic DNA polymerases such as the Klenow fragment of DNA polymerase I, T4 DNA polymerase and Taq polymerase, the DNA metabolic enzyme DNase I, and even a DNA polymerase from a higher plant, cauliflower. Similarly, the compounds did not inhibit the activity of the human immunodeficiency virus type 1 reverse transcriptase. The kinetic studies of the compounds showed that DNA polymerase α was inhibited non-competitively with respect to the DNA template and substrate, whereas DNA polymerase β was inhibited competitively with both the DNA template and substrate. The binding to DNA polymerase β could be stopped with non-ionic detergent, but the binding to DNA polymerase α could not.

Effect of triphosphate modifications in 2′-deoxynucleoside 5′-triphosphates on their specificity towards various DNA polymerases

Some natural and glycon-modified dNTPs with β,γ-pyrophosphate substitution at the triphosphate residue were synthesized and studied to evaluate the effect of these modifications on substrate properties of dNTPs in DNA synthesis catalyzed by human placental DNA polymerases α and β, avian myeloblastosis virus reverse transcriptase, and calf thymus terminal deoxynucleotidyl transferase. Reverse transcriptase proved to be the enzyme least specific to such modifications; the substrate activity of β,γ-methylenediphosphonate substituted dTTP and 3′-azido-3′-deoxy-dTTP decreased in the following order: CF 2=CHF>CBr 2>CFMe≫CH 2. This order is individual for each DNA polymerase. It is interesting to mention that β,γ-CBr 2 substituted dTTP is neither a substrate nor an inhibitor of DNA polymerase β. This specificity distinguishes DNA polymerase β from other DNA polymerases studied.

Stereoisomers of Deoxynucleoside 5′-Triphosphates as Substrates for Template-dependent and -independent DNA Polymerases

All four possible stereoisomers of dNTP with regard to deoxyribofuranose C-1' and C-4' carbon atoms were studied as substrates for several template-dependent DNA polymerases and template-independent terminal deoxynucleotidyl transferase. It was shown that DNA polymerases alpha, beta, and epsilon from human placenta and reverse transcriptases of human immunodeficiency virus and avian myeloblastosis virus incorporate into the DNA chain only natural beta-D-dNTPs, whereas calf thymus terminal deoxynucleotidyl transferase incorporates two nucleotide residues of alpha-D-dNTP and extends the resulting oligonucleotide in the presence of beta-D-dNTPs. The latter enzyme also extended alpha-anomeric D-oligodeoxynucleotide primers in the presence of beta-D-dNTPs. None of the studied enzymes utilized L-dNTPs. These data indicate that template-dependent DNA polymerases are highly stereospecific with regard to dNTPs, whereas template-independent terminal deoxynucleotidyl transferase shows less stereodifferentiation. It is likely that the active center of the latter enzyme forms no specific contacts with the nucleic bases of both nucleotide substrate and oligonucleotide primer.

4′-Thio-5-Ethyl-2′-Deoxyuridine5′-Triphosphate (TEDUTP): Synthesis and Substrate Properties in DNA-Synthesizing Systems

An efficient synthesis of the 5′-phosphate and 5′-triphosphate of a 4′-thio-2′-deoxynucleoside is described for the first time. The 5′-triphosphate of the antiherpesvirus agent 4′-thio-5-ethyl-2′-deoxyuridine (TEDU) has been shown to be a good substrate for human placental DNA polymerase α, calf thymus terminal deoxynucleotidyl transferase, and the reverse transcriptases of human immunodeficiency virus and avian myeloblastosis virus. As it is known that TEDU is a substrate only for herpesvirus-encoded thymidine kinases and not the cellular kinases, it is likely that the utilization of TEDUTP by DNA-synthesizing enzymes results in the selective toxicity seen in vitro in herpesvirus-infected cells.

γ-Phosphate-substituted 2′-Deoxynucleoside 5′-Triphosphates as Substrates for DNA Polymerases

Several 2'-deoxythymidine 5'-triphosphate and 3'-azido-2', 3'-dideoxythymidine 5'-triphosphate analogs containing a hydrophobic phosphonate group instead of the gamma-phosphate were synthesized and evaluated as substrates for human immunodeficiency virus (HIV) and avian myeloblastosis virus reverse transcriptases, human placental DNA polymerases alpha and beta, and calf thymus terminal deoxynucleotidyl transferase. They were efficiently incorporated into the DNA chain by the retroviral enzymes but were not utilized by the mammalian ones. Also, some gamma-ester and gamma-amide derivatives of dTTP and 3'-azido-2',3'-dideoxythymidine 5'-triphosphate (AZTTP) were synthesized and studied. They proved to be substrates for both the retroviral and mammalian enzymes under study. The Km values for incorporation of the dTTP derivatives into the DNA chain were close to those for dTTP and AZTTP. The Km for the AZTTP derivatives were one order of magnitude greater than those for dTTP and AZTTP. The results obtained indicate that HIV and avian myeloblastosis virus reverse transcriptases have no sterical obstacles for binding the triphosphate fragment bearing a bulky substituent at the gamma-position. Modification of the gamma-phosphate in AZTTP increased the selectivity of HIV reverse transcriptase inhibition versus DNA polymerase alpha. gamma-Methylphosphonate and gamma-phenylphosphonate were dephosphorylated in human serum much less rapidly than AZTTP. Besides, they were shown to be markedly more hydrophobic than AZTTP. Thus, replacement of the gamma-phosphate in AZTTP with gamma-phosphonate markedly alters its substrate properties toward some cellular DNA polymerases and blood dephosphorylating enzymes but does not change its substrate activity with respect to HIV reverse transcriptase.

Effect of 3'-amino-2',3'-dideoxycytidine on DNA replicative intermediates

3'-Amino-2',3'-dideoxycytidine (3'-NH 2-ddCyd) is a 3'-modified deoxycytidine analog that specifically inhibits DNA synthesis. Inhibition of chain elongation at the replication fork was examined utilizing a batch hydroxylapatite chromatography method. Exponentially growing cells were exposed to 3'-NH 2-ddCyd and the diterpene aphidicolin for 9.5 hr at concentrations that inhibited DNA synthesis by approximately 60 and 90%, as determined by precursor uptake. Both agents demonstrated a concentration-dependent inhibition of pulse labeling of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) generated by a limited alkaline lysis procedure. Upon removal of drug, the rate of elongation of pulse-labeled DNA was similar to that of untreated cells at both concentrations of aphidicolin and at the low concentration of the amino analog. Under these conditions, no reduction in cell survival was observed using the clonogenic assay technique. However, at the high concentration of 3'-NH 2-ddCyd, the rate of elongation following drug removal was one-third that of untreated cultures, and a 50% loss in cell viability was observed. Furthermore, upon incubation of purified dsDNA with the Klenow fragment of Escherichia coli DNA polymerase I or purified ssDNA with calf thymus terminal deoxynucleotidyl transferase, only DNA from cells treated with the high concentration of 3'-NH 2-ddCyd served as a poor template for further synthesis. The results indicate that 3'-NH 2-ddCyd, in a concentration-dependent manner, inhibits DNA synthesis by reducing the rate of chain elongation at the replication fork, which subsequently leads to a functional blocking of 3'-ends in DNA. The data suggest that there may be a relationship between loss of cell viability and reduction in the number of 3'-ends available for DNA replication.

A novel method for site specific introduction of single model oxidative DNA lesions into oligodeoxyribonucleotides.

Calf thymus terminal deoxynucleotidyl transferase was used to incorporate several products of oxidative base damage onto the 3' end of oligodeoxyribonucleotides. Under the defined conditions described in this report, single residues of dihydrothymine, beta-ureidoisobutyric acid, thymine glycol, urea, 7-hydro-8-oxoadenine, 7-hydro-8-oxoguanine, 5-hydroxycytosine and 5-hydroxyuracil were incorporated into oligodeoxyribonucleotides of different lengths. The reaction is both efficient and cost effective. The 3' termini of the reaction products were suitable substrates for ligation by phage T4 DNA ligase, facilitating greatly the construction of oligodeoxyribonucleotides containing unique and site specific oxidative DNA lesions.

Properties of 2′,3′-dideoxy-2′,3′-dehydrothymidine 5′-triphosphate in terminating DNA synthesis catalyzed by several different DNA polymerases

2′-3′-Dideoxy-2′,3′-dehydrothymidine 5′-triphosphate (dddTTP) shows termination substrate properties in the DNA synthesis catalyzed by E. coli DNA polymerase I KF, rat liver DNA polymerase β, reverse transcriptases of avian myeloblastosis virus and Raus sarcoma virus and calf thymus terminal deoxynucleotidyl transferase. This implies that the mononucleotide residue of dddTTP incorporates into 3′-termini of newly synthesized DNA chains. However, dddTTP has no influence on the DNA synthesis catalyzed by calf thymus DNA polymerase α. In the case of some DNA polymerases dddTTP was one order of magnitude more effective in comparison with the other known termination substrates.

Biochemistry of terminal deoxynucleotidyltransferase (TdT): characterization and mechanism of inhibition of TdT by P1,P5-bis(5'-adenosyl) pentaphosphate

The catalysis of DNA synthesis by calf thymus terminal deoxynucleotidyltransferase (TdT) is strongly inhibited in the presence of Ap5A, while replicative DNA polymerases from mammalian, bacterial, and oncornaviral sources are totally insensitive to Ap5A addition. The Ap5A-mediated inhibition of TdT seems to occur via its interaction at both the substrate binding and primer binding domains as judged by classical competitive inhibition plots with respect to both substrate deoxynucleoside triphosphate (dNTP) and DNA primer and inhibition of ultraviolet light mediated cross-linking of substrate dNTP and oligomeric DNA primer to their respective binding sites. Further kinetic analyses of Ap5A inhibition revealed that the dissociation constant of the Ap5A-enzyme complex, with either substrate binding or primer binding domain participating in the complex formation, is approximately 6 times higher (Ki = 1.5 microM) compared to the dissociation constant (Ki = 0.25 microM) of the Ap5A-TdT complex when both domains are available for binding. In order to study the binding stoichiometry of Ap5A to TdT, an oxidized derivative of Ap5A, which exhibited identical inhibitory properties as its parent compound, was employed. The oxidation product of Ap5A, presumably a tetraaldehyde derivative, binds irreversibly to TdT when the inhibitor-enzyme complex is subjected to borohydride reduction. The presence of aldehyde groups in the oxidized Ap5A appeared essential for inhibitory activity since its reduction to alcohol via borohydride reduction or its linkage to free amino acids prior to use as an inhibitor rendered it completely ineffective.(ABSTRACT TRUNCATED AT 250 WORDS)