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

Open AccessPublished:April 04, 1997DOI:https://doi.org/10.1074/jbc.272.14.9556
      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 α, β, and ϵ from human placenta and reverse transcriptases of human immunodeficiency virus and avian myeloblastosis virus incorporate into the DNA chain only natural β-D-dNTPs, whereas calf thymus terminal deoxynucleotidyl transferase incorporates two nucleotide residues of α-D-dNTP and extends the resulting oligonucleotide in the presence of β-D-dNTPs. The latter enzyme also extended α-anomeric D-oligodeoxynucleotide primers in the presence of β-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.

      INTRODUCTION

      The substrate activity of the stereoisomers of DNA polymerase natural substrates and their analogs is of significant current interest, because these compounds can help to ascertain the mechanism of substrate binding by DNA polymerases and identify the parts of the dNTP molecule that specifically bind to the active center of the enzymes. Indeed, the stereoisomers of natural β-D-dNTPs
      The abbreviations used are: β-D-dNTP
      natural 2′-deoxynucleoside 5′-triphosphate
      α-D-dNTP with N being C, T, or A
      α-anomer at C-1′ carbon of dNTP
      β-L-dNTP with N being C or T
      β-D-dNTP enantiomer
      α-L-dNTP with N being C, T, or A
      α-anomer at C-1′ carbon of β-L-dNTP, β-D-dNMP, natural 2′-deoxynucleoside 5′-monophosphate
      α-D-dNMP
      α-anomer at C-1′ carbon of dNMP, β-L-dTMP, β-D-dTMP enantiomer
      TDT
      terminal deoxynucleotidyl transferase
      HIV
      human immunodeficiency virus
      HPLC
      high pressure liquid chromatography.
      differ only in the mutual orientation of the reaction center (triphosphate fragment), the genetic recognition element (nucleic base), and the sugar residue.
      Recently β-L-dTTP has been shown to be an inhibitor of HIV reverse transcriptase (
      • Yamaguchi T.
      • Iwanami N.
      • Shudo K.
      • Saneyoshi M.
      ). It inhibited elongation of oligo(dT)12-18 complexed with poly(rA), by 50% at a [β-L-dTTP]/[dTTP] concentration ratio of 0.1. Although it was not demonstrated by direct experiments, such a strong inhibitory effect is most likely to result from chain termination. The inhibitory effect of β-L-dTTP on mammalian mitochondrial DNA polymerase γ was weaker than that observed for HIV reverse transcriptase. DNA polymerases α and β did not utilize β-L-dTTP as a substrate (
      • Yamaguchi T.
      • Iwanami N.
      • Shudo K.
      • Saneyoshi M.
      ). Focher et al. (
      • Focher F.
      • Maga G.
      • Bendiscioli A.
      • Capobianco M.
      • Colonna F.
      • Garbesi A.
      • Spadari S.
      ) have studied the substrate properties of β-L-dTTP toward human DNA polymerases α, β, γ, δ, and ϵ, as well as herpes simplex virus type 1 DNA polymerase, Escherichia coli DNA polymerase I, HIV reverse transcriptase, and TDT using poly(dA)·oligo(dT)20 as a template-primer for template-dependent enzymes and oligo(dT)20 as a primer for TDT. DNA polymerases β, γ, δ, and ϵ did not incorporate nucleotide residues of this compound into the DNA chain, whereas the other enzymes extended the primer by one or more β-L-dTMP residues. Specifically, DNA polymerases α and Klenow fragment incorporated two β-L-dTMP residues, and HIV reverse transcriptase elongated the primer by up to 3 or 4 β-L-dTMP residues.
      Furthermore, it has been shown (
      • Van Draanen N.A.
      • Tucker S.C.
      • Boyd F.L.
      • Trotter B.W.
      • Reardon J.E.
      ) that 2′,3′-dideoxy-β-L-thymidine 5′-triphosphate and 2′,3′-dideoxy-2′,3′-didehydro-β-L-thymidine 5′-triphosphate are incorporated into DNA chains by HIV reverse transcriptase, E. coli DNA polymerase I, and T7 DNA polymerase, but their affinity to the HIV enzyme is 10-50 times lower than that of their β-D-isomers. The β-L-stereoisomers of 2′,3′-dideoxy-2′,3′-didehydrocyclopentane-adenine 5′-α-methylenephosphonyl-β,γ-diphosphate (
      • Semizarov D.G.
      • Victorova L.S.
      • Dyatkina N.B.
      • von Janta-Lipinsky M.
      • Krayevsky A.A.
      ) and its guanine counterpart (
      • Merlo V.
      • Roberts S.M.
      • Storer R.
      • Bethell R.C.
      ), as well as several (−)-β-L-oxathiolanenucleoside 5′-triphosphates (
      • Hart G.J.
      • Orr D.C.
      • Penn C.R.
      • Figueiredo H.T.
      • Gray N.M.
      • Boehme R.E.
      • Cameron J.M.
      ,
      • Chang C.-N.
      • Skalski V.
      • Zhou J.H.
      • Cheng Y.-C.
      ,
      • Skalski V.
      • Chang C.-N.
      • Dutschman G.
      • Cheng Y.-C.
      ,
      • Wilson J.E.
      • Martin J.L.
      • Borroto-Esoda K.
      • Hopkins S.
      • Painter G.
      • Liotta D.C.
      • Furman P.A.
      ) and (−)-β-L-dioxolanenucleoside 5′-triphosphates (
      • Kukhanova M.
      • Liu S.-H.
      • Mozzherin D.
      • Lin T.-S.
      • Chu C.K.
      • Cheng Y.-C.
      ), have been shown to be terminating substrates for a number of DNA polymerases.
      To date, no compounds have been found that would specifically inhibit TDT in cell cultures. One of the reasons for that is the similarity in substrate specificity between TDT and some other DNA polymerases, especially DNA polymerase β (
      • Beabealashvilli R.S.
      • Scamrov A.V.
      • Kutateladze T.V.
      • Mazo A.M.
      • Krayevsky A.A.
      • Kukhanova M.K.
      ,
      • Krayevsky A.A.
      • Kukhanova M.K.
      ) and endogeneous reverse transcriptases (
      • Pyrinova G.B.
      • Kuzminova E.A.
      • Salganik R.I.
      • Krayevsky A.A.
      • Kukhanova M.K.
      ).
      However, the independence of TDT from the template is a factor that could simplify the design of selective inhibitors of this enzyme. Indeed, we have recently found (
      • Semizarov D.G.
      • Victorova L.S.
      • Dyatkina N.B.
      • von Janta-Lipinsky M.
      • Krayevsky A.A.
      ) that dNTP analogs with trans-like orientation of the nucleic base and triphosphate residue efficiently and selectively inhibit DNA synthesis catalyzed by TDT.
      In this paper we synthesized all four possible stereoisomers of dNTPs with respect to the C-1′ and C-4′ carbon atoms and evaluated them as substrates for template-dependent DNA polymerases α, β, and ϵ from human placenta, reverse transcriptases from HIV and avian myeloblastosis virus, and TDT from calf thymus. We also synthesized two anomeric α-D-oligodeoxynucleotides and studied them as primers for TDT.

      MATERIALS AND METHODS

      The starting compounds, β-L-, α-D- and α-L-2′-deoxynucleosides, were synthesized as described in Refs.
      • Spadari S.
      • Maga G.
      • Focher F.
      • Ciarrocchi G.
      • Manservigi R.
      • Arcamone F.
      • Capobianco M.
      • Carcuro A.
      • Colonna F.
      • Iotti S.
      • Garbesi A.
      ,
      • Yamaguchi T.
      • Saneyoshi M.
      and
      • Génu-Dellac C.
      • Gosselin G.
      • Puech F.
      • Henry J.-C.
      • Aubertin A.-M.
      • Obert G.
      • Kirn A.
      • Imbach J.-L.
      respectively. 2′-Deoxynucleoside 5′-triphosphates were purchased from Boehringer Mannheim. α-d[(Tp)3T] and α-d[(Tp)11)T] were synthesized according to
      • Morvan F.
      • Rayner B.
      • Leonetti J.-P.
      • Imbach J.-L.
      ; β-d[(Tp)3T] was generously provided by Dr. T. Bocharova (Institute of Molecular Genetics, Moscow, Russia).
      β-L-dNTPs, α-D-dNTPs, and α-L-dNTPs (Fig. 1) were synthesized according to Ludwig (
      • Ludwig J.
      ) using POCl3 and pyrophosphate. For purification of dNTP stereoisomers, DEAE-Toyopearl 650 M (Toyosoda) and LiChroprep RP-18 (40-63 μm, Merck) were used. Their UV characteristics are listed in Table I. All stereomers were separated from possible natural dNTP contaminations by HPLC; the retention times are given in Table I.
      Figure thumbnail gr1
      Fig. 1Stereoisomers of deoxynucleoside 5′-triphosphates.
      TABLE ICharacteristics of dNTP stereoisomers
      CompoundUV spectrum, λmaxRetention time
      nmmin
      β-L-dTTP268 (pH 7)10.5
      β-L-dCTP275 (pH 10)6.5
      281 (pH 2)
      α-D-dATP261 (pH 7)12.5 (12.8)
      α-D-dCTP275 (pH 10)7 (6.5)
      281 (pH 2)
      α-D-dTTP268 (pH 7)11.5 (10.5)
      α-L-dATP261 (pH 7)12.5
      α-L-dCTP275 (pH 10)7
      281 (pH 2)
      α-L-dTTP268 (pH 7)11.5

      Enzymes and DNA

      HIV reverse transcriptase was isolated according to
      • Rozovskaya T.A.
      • Belogurov A.A.
      • Lukin M.A.
      • Chernov D.N.
      • Kukhanova M.K.
      • Bibilashvili R.S.
      . DNA polymerases α and ϵ were isolated from human placenta as described in
      • Mozzherin D.J.
      • Atrazhev A.M.
      • Kukhanova M.K.
      ; DNA polymerase β was purified according to
      • Kolocheva T.A.
      • Nevinsky G.A.
      . Avian myeloblastosis virus reverse transcriptase and calf thymus TDT were from Omutninsk Chemicals (Russia) and Amersham Corp., respectively.
      Single-stranded M13mp10 DNA was isolated from the culture medium of the recipient E. coli K12XL1 strain as described in
      • Kraev A.A.
      . Tetradecanucleotide primers a and b (Fig. 2) and α-D-oligonucleotides were labeled at the 5′ terminus using [γ-32P]ATP (Radioizotop, Russia) and T4 polynucleotide kinase (Amersham Corp.) according to
      • Sambrook J.
      • Fritsch E.F.
      • Maniatis T.
      . The DNA (0.5 μM) was hybridized with 0.75 μM [5′-32P]-labeled primer in the following buffers: 10 mM Tris-HCl (pH 8.2), 5 mM MgCl2, 40 mM KCl, and 1 mM dithiothreitol (for reverse transcriptases); 10 mM Tris-HCl (pH 7.4), 6 mM MgCl2, and 0.4 mM dithiothreitol (for DNA polymerase α); and 10 mM Tris-HCl (pH 8.5), 5 mM MgCl2, and 1 mM dithiothreitol (for DNA polymerase β).
      Figure thumbnail gr2
      Fig. 2Structure of the template-primers.

      Primer Extension Assays

      For the template-dependent DNA polymerases, the assay mixture (volume, 6 μl) contained 0.01 μM template-primer (Fig. 2), stereoisomer under study or its natural counterpart, enzyme (2 activity units of reverse transcriptase or 1 unit of DNA polymerases α and β), and the corresponding buffer. The reaction was carrried out for 20 min at 37°C and terminated by adding 3 μl of deionized formamide containing 0.5 mM EDTA and 2% bromphenol blue and xylene cyanol. The reaction products were separated by electrophoresis in 20% polyacrylamide gel, and the gels obtained were autoradiographed.
      For TDT, the assay mixture (volume, 5 μl) contained 0.1 μM 5′-32P-labeled tetradecanucleotide primer (Fig. 2) or α-D-oligonucleotide, compound under study or natural dNTP, 2 units of the enzyme, 100 mM sodium cacodylate (pH 7.2), 10 mM MgCl2, 1 mM CoCl2, and 1 mM 2-mercaptoethanol.

      RESULTS

      The structure of the dNTP stereoisomers studied is shown in Fig. 1.
      The fidelity of DNA synthesis catalyzed by HIV reverse transcriptase is rather low (
      • Takeuchi Y.
      • Nagumo T.
      • Hoshino H.
      ,
      • Preston B.D.
      • Poiesz B.J.
      • Loeb L.A.
      ,
      • Roberts J.D.
      • Bebenek K.
      • Kunkel T.A.
      ), and the probability of incorrect dNMP incorporation is higher for the template positions remote from the primer 3′ terminus by more than two nucleotide residues. Therefore, we used different template-primers for the dCTP, dTTP, and dATP stereoisomers (type of template-primer used is specified in figure caption).
      It is evident from Fig. 3 that β-L-dCTP is not incorporated into the DNA chain by HIV reverse transcriptase (Fig. 3A, lanes 4-6). In the control assays, dATP (Fig. 3A, lane 2) and dATP + dCTP (Fig. 3A, lane 3) were used. Similar results were obtained for avian myeloblastosis virus reverse transcriptase (data not shown). We also evaluated β-L-dCTP and β-L-dTTP as substrates for human DNA polymerases α, β, and ϵ. It can be seen in Fig. 4 that β-L-dCTP is also not incorporated into the DNA chain by these enzymes (Fig. 4 A and B, lanes 4-6). It was not a substrate for DNA polymerase ϵ (data not shown).
      Figure thumbnail gr3
      Fig. 3Series A, primer extension catalyzed by HIV reverse transcriptase. Lane 1, template-primer a + enzyme; lane 2, as in lane 1 + 2 μM dATP; lane 3, as in lane 1 + 2 μM dATP + 2 μM dCTP; lanes 4 and 5, as in lane 1 + 2 μM dATP + 2 μM (lane 4) and 20 μM (lane 5) β-L-dCTP; lane 6, as in lane 1 + 2 μM dATP + 20 μMβ-L-dCTP + 20 μM dGTP. Series B, primer extension catalyzed by TDT. Lane 1, primer a + enzyme; lanes 2 and 3, as in lane 1 + 0.2 μM (lane 2) and 2 μM (lane 3) dCTP; lanes 4-6, as in lane 1 + 0.2 μM (lane 4), 2 μM (lane 5), and 20 μM (lane 6) β-L-dCTP.
      Figure thumbnail gr4
      Fig. 4Primer extension by DNA polymerases α (A) and β (B). Lanes 1, template-primer a + enzyme; lanes 2, as in lanes 1 + 2 μM dATP; lanes 3, as in lanes 1 + 2 μM dATP + 2 μM dCTP; lanes 4 and 5, as in lanes 1 + 2 μM dATP + 2 μM (lanes 4) and 20 μM (lanes 5) β-L-dCTP; lanes 6, as in lanes 1 + 2 μM dATP + 20 μMβ-L-dCTP + 20 μM dGTP.
      Fig. 3B presents the results of TDT assays in the presence of β-L-dCTP. Clearly, some incorporation is observed at 2 (Fig. 3B, lane 5) and 20 (Fig. 3B, lane 6) μM, but scanning densitometry revealed that the extent of primer conversion is only 2% at 20 μM. The β-L-dTTP displayed the same substrate activity as β-L-dCTP for all DNA polymerases studied (data not shown).
      It can be seen in Fig. 5 that α-L-dNTPs (lanes 3, 4, 6, and 7) and α-D-dNTPs (lanes 8, 9, 11, and 12) are not substrates for HIV reverse transcriptase. We assume that α-L-dATP practically does not interact with the DNA-synthesizing complex, because it did not inhibit primer extension by dTMP and dGMP residues even at a high concentration (Fig. 5 lanes 6-7), whereas α-D-dATP completely inhibited primer extension at a [α-D-dATP]/[dNTP] concentration ratio of 10:1 (Fig. 5 lane 12). We attribute the presence of a weak heptadecanucleotide band in lane 2 of Fig. 5 to the error-prone properties of HIV reverse transcriptase. Both α-D-dNTPs and α-L-dNTPs were not utilized as substrates by human DNA polymerases (data not shown).
      Figure thumbnail gr5
      Fig. 5Primer extension catalyzed by HIV reverse transcriptase. Lane 1, template-primer b + enzyme; lane 2, as in lane 1 + 2 μM dTTP; lanes 3 and 4, as in lane 1 + 2 μM (lane 3) and 20 μM (lane 4) α-L-dTTP; lane 5, as in lane 1 + 2 μM dTTP + 2 μM dGTP; lanes 6 and 7, as in lane 1 + 2 μM dTTP + 2 μM dGTP + 2 μM (lane 6) and 20 μM (lane 7) α-L-dATP; lanes 8 and 9, as in lane 1 + 2 μM (lane 8) and 20 μM (lane 9) α-D-dTTP; lane 10, as in lane 1 + 2 μM dTTP + 2 μM dGTP; lanes 11 and 12, as in lane 1 + 2 μM dTTP + 2 μM dGTP + 2 μM (lane 11) and 20 μM (lane 12) α-D-dATP.
      It is evident from Fig. 6 that two α-D-dTMP (lanes 10-12) and α-D-dAMP (lanes 13-15) residues are incorporated into the primer by TDT, and one more residue is incorporated less efficiently. The efficiency of primer extension depended on the substrate concentration. Similar results were obtained for α-D-dCTP (data not shown). The α-L-dTTP (Fig. 6 lanes 4-6) and α-L-dATP were not utilized by the enzyme. In the control assays (Fig. 6 lanes 2 and 3), dTTP was used as a substrate. We ascribe the presence of weak pentadecanucleotide bands on lanes 1, 5, and 6 of Fig. 6 to contamination of the TDT preparation with trace amounts of dNTPs.
      Figure thumbnail gr6
      Fig. 6Primer extension catalyzed by TDT. Lane 1, Primer b + enzyme; lanes 2 and 3, as in lane 1 + 0.2 μM (lane 2) and 2 μM (lane 3) dTTP; lanes 4-6, as in lane 1 + 0.2 μM (lane 4), 2 μM (lane 5), and 20 μM (lane 6) α-L-dTTP; lanes 7-9, as in lane 1 + 0.2 μM (lane 7), 2 μM (lane 8), and 20 μM (lane 9) α-L-dATP; lanes 10-12, as in lane 1 + 0.2 μM (lane 10), 2 μM (lane 11), and 20 μM (lane 12) α-D-dTTP; lanes 13-15, as in lane 1 + 0.2 μM (lane 13), 2 μM (lane 14), and 20 μM (lane 15) α-D-dATP.
      Then, we examined the ability of TDT to elongate oligonucleotides terminated by α-D-dNMP residues in the presence of different concentrations of dNTPs. It can be seen in Fig. 7 that oligodeoxynucleotides containing α-D-dTMP and α-D-dAMP residues at the 3′ end are elongated by TDT in the presence of 0.2, 2, and 20 μM dNTPs (lanes 6-8 and 11-13), the length of the products being dependent on the dNTP concentration.
      Figure thumbnail gr7
      Fig. 7Primer extension catalyzed by TDT. Lane 1, primer b + enzyme; lanes 2 and 3, as in lane 1 + 0.2 μM (lane 2) and 2 μM (lane 3) dTTP; lanes 4-8, as in lane 1 + 2 μM (lane 4) and 20 μM (lanes 5-8) α-D-dTTP; lanes 9-13, as in lane 1 + 2 μM (lane 9) and 20 μM (lanes 10-13) α-D-dATP. In lanes 6-8 and 11-13, 0.2 μM (lanes 6 and 11), 2 μM (lanes 7 and 12), and 10 μM (lanes 8 and 13) dNTPs were added after 20 min of incubation, and the mixture was incubated for a further 20 min.
      At the second stage of this research we evaluated α-d[(Tp)11T] and α-d[(Tp)3T] as primers for TDT in the presence of natural dNTPs and α-dNTP (Fig. 8 B and D). Oligonucleotides β-d[(Tp)9T] (Fig. 8A) and β-d[(Tp)3T] (Fig. 8C) were used as reference primers. All four oligothymidylates were extended in the presence of dGTP, and the length of the products depended on the dGTP concentration (Fig. 8 lanes 2-4). Similar results were obtained with dCTP as substrate.
      Figure thumbnail gr8
      Fig. 8Extension of β-d[(Tp)9T] (A), α-d[(Tp)11T] (B), β-d[(Tp)3T] (C), and α-d[(Tp)3T] (D) catalyzed by TDT. Lanes 1, oligodeoxythymidylate + enzyme; lanes 2-4, as in lanes 1 + 0.2 μM (lanes 2), 2 μM (lanes 3), and 20 μM (lanes 4) dGTP.

      DISCUSSION

      Our results indicate that template-dependent DNA polymerases utilize as substrates only β-D-dNTPs, whereas for template-independent TDT α-D-dNTPs but not β-L-dNTPs or α-L-dNTPs are substrates. It is noteworthy that α-D-dNTPs but not β-L-dNTPs or α-L-dNTPs inhibited primer extension catalyzed by HIV and avian myeloblastosis virus reverse transcriptases. It seems likely that the 3′ hydroxyl of L-dNTPs hinders formation of the productive [DNA polymerase + template-primer + dNTP] complex by creating steric obstacles for dNTP binding to the enzyme.
      The inconsistency between our results and the data of Focher et al. (
      • Focher F.
      • Maga G.
      • Bendiscioli A.
      • Capobianco M.
      • Colonna F.
      • Garbesi A.
      • Spadari S.
      ) may be attributed to the following differences in the experimental conditions. First, these authors used a homopolymeric template-primer, whereas in our experiments random-sequence tetradecanucleotides and M13mp10 DNA were employed. Thus, our system better models the natural conditions of DNA biosynthesis. Second, Focher et al. used groundlessly high concentrations of β-L-dTTP (up to 0.5-1 mM). Obviously, this may initiate various side processes. When we repeated our experiments using these high concentrations of β-L-dNTPs, many uninterpretable bands were observed. Finally, formation of oligo(dT)19 observed by Focher et al. (
      • Focher F.
      • Maga G.
      • Bendiscioli A.
      • Capobianco M.
      • Colonna F.
      • Garbesi A.
      • Spadari S.
      ) upon primer extension by DNA polymerase α, HIV reverse transcriptase, and TDT (Fig. 5 in Ref. 2) may result only from pyrophosphorolysis of oligo(dT)20, because these enzymes do not possess 3′→ 5′ exonuclease activity. Indeed, ion-exchange chromatography performed in
      • Focher F.
      • Maga G.
      • Bendiscioli A.
      • Capobianco M.
      • Colonna F.
      • Garbesi A.
      • Spadari S.
      cannot properly separate inorganic pyrophosphate from β-L-dTTP, because these compounds have close charges under the conditions described. Thus, it is possible that pyrophosphorolysis led to formation of β-D-dTTP in the assay mixture and subsequent primer extension. Therefore, for our part, we additionally purified β-L-dTTP and β-L-dCTP by reversed-phase HPLC.
      Interestingly, the affinity of 3′-modified β-L-dNTP analogs to human DNA polymerases α, β, ϵ, and γ and HIV reverse transcriptase drops as the bulk of the substituent is increased, O > S > CH ≈ CH2 > CH2OH (Refs.
      • Chang C.-N.
      • Skalski V.
      • Zhou J.H.
      • Cheng Y.-C.
      and
      • Kukhanova M.
      • Liu S.-H.
      • Mozzherin D.
      • Lin T.-S.
      • Chu C.K.
      • Cheng Y.-C.
      ; this paper for human DNA polymerases; Refs.
      • Van Draanen N.A.
      • Tucker S.C.
      • Boyd F.L.
      • Trotter B.W.
      • Reardon J.E.
      ,
      • Hart G.J.
      • Orr D.C.
      • Penn C.R.
      • Figueiredo H.T.
      • Gray N.M.
      • Boehme R.E.
      • Cameron J.M.
      and
      • Wilson J.E.
      • Martin J.L.
      • Borroto-Esoda K.
      • Hopkins S.
      • Painter G.
      • Liotta D.C.
      • Furman P.A.
      for HIV reverse transcriptase). It is possible that the dNTP-binding site of DNA polymerases contains one or several groups near the C-3′ atom of β-L-dNTP, which hinders productive dNTP binding.
      It has earlier been shown (
      • Semizarov D.G.
      • Victorova L.S.
      • Dyatkina N.B.
      • von Janta-Lipinsky M.
      • Krayevsky A.A.
      ) that carbocyclic α-D- and L-dNTP analog isosteres I and II (Fig. 9) are incorporated into the DNA chain by TDT, but are not utilized by template-dependent DNA polymerases. In this work we showed that α-D-dNTPs are substrates for TDT but are not recognized by template-dependent DNA polymerases, suggesting that they could be used as selective inhibitors of TDT to elucidate the functional role of this enzyme. On the other hand, unlike their carbocyclic counterpart II, α-L-dNTPs were not incorporated into the primer by TDT. We ascribe this difference in substrate properties to the presence of a double C-2′-C-3′ bond in II. Indeed, the latter is known to impart planarity to the sugar moiety and drastically increase the affinity of the dNTP to DNA polymerases (
      • Semizarov D.G.
      • Victorova L.S.
      • Dyatkina N.B.
      • von Janta-Lipinsky M.
      • Krayevsky A.A.
      ,
      • Dyatkina N.
      • Minassyan S.
      • Kukhanova M.
      • Krayevsky A.A.
      • von Janta- Lipinski M.
      • Chidgeavadze Z.
      • Beabealashvilli R.
      ,
      • Krayevsky A.A.
      • Watanabe K.A.
      ). It is likely that α-L-dNTPs do not interact productively with TDT because of the difference in the position of the 3′-hydroxyl in L- and D-dNTPs.
      Figure thumbnail gr9
      Fig. 9Structure of the TDT substrate carbocyclic α-D- and L-dNTP analogs I and II and the TDT inhibitor phenyl phosphonyldiphosphate III.
      Because β-oligodeoxyribonucleotides containing one or two α-D-dNMP residues at the 3′ terminus are extended by TDT in the presence of natural dNTPs (Fig. 7), it seemed interesting to evaluate oligodeoxyribonucleotides containing only α-D-dNMP residues as a primer for TDT. Another goal at this stage of the research was to find oligonucleotides utilized as primers by TDT and stable in the cell and blood, i.e. resistant toward nucleases. Short α-oligonucleotides are interesting in this respect due to their increased stability in serum (
      • Debart F.
      • Tosquellas G.
      • Rayner B.
      • Imbach J.-L.
      ). In this work we compared two α-oligothymidylates with their β counterparts as primers for TDT and found that all four oligonucleotides are extended by this enzyme, although the α-oligothymidylates are slightly less efficient as primers (Fig. 8).
      The results obtained may imply that the nucleic bases of dNTP and oligonucleotide primers do not bind in a specific manner to the substrate- and primer-binding sites, respectively, of the TDT active center. Indeed, we have found that phenylphosphonyldiphosphate (III, Fig. 9) was not a substrate for TDT (data not shown), but inhibited TDT-catalyzed primer extension by 50% at a [III]/[dTTP] molar concentration ratio of 1:1.

      Acknowledgments

      We are grateful to Dr. L. Victorova (Engelhardt Institute of Molecular Biology, Moscow, Russia) for providing DNA polymerase β and to Drs S. Spadari and F. Focher (Instituto di Genetica Biochimica ed Evoluzionistica, Pavia, Italy) for useful discussion.

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