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

(Received for publication, November 5, 1996)

Dmitry G. Semizarov Dagger , Andrey A. Arzumanov Dagger , Natalya B. Dyatkina Dagger , Albert Meyer §, Sophie Vichier-Guerre §, Gilles Gosselin §, Bernard Rayner §, Jean-Louis Imbach § and Alexander A. Krayevsky Dagger

From the Dagger  Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov Street, Moscow, 117984 Russia and the § Laboratoire de Chimie Bioorganique, CNRS UMR 5625, Université de Montpellier II, Place Eugene Bataillon, 34095 Montpellier Cedex 5, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

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.


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 beta -D-dNTPs1 differ only in the mutual orientation of the reaction center (triphosphate fragment), the genetic recognition element (nucleic base), and the sugar residue.

Recently beta -L-dTTP has been shown to be an inhibitor of HIV reverse transcriptase (1). It inhibited elongation of oligo(dT)12-18 complexed with poly(rA), by 50% at a [beta -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 beta -L-dTTP on mammalian mitochondrial DNA polymerase gamma  was weaker than that observed for HIV reverse transcriptase. DNA polymerases alpha  and beta  did not utilize beta -L-dTTP as a substrate (1). Focher et al. (2) have studied the substrate properties of beta -L-dTTP toward human DNA polymerases alpha , beta , gamma , delta , and epsilon , 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 beta , gamma , delta , and epsilon  did not incorporate nucleotide residues of this compound into the DNA chain, whereas the other enzymes extended the primer by one or more beta -L-dTMP residues. Specifically, DNA polymerases alpha  and Klenow fragment incorporated two beta -L-dTMP residues, and HIV reverse transcriptase elongated the primer by up to 3 or 4 beta -L-dTMP residues.

Furthermore, it has been shown (3) that 2',3'-dideoxy-beta -L-thymidine 5'-triphosphate and 2',3'-dideoxy-2',3'-didehydro-beta -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 beta -D-isomers. The beta -L-stereoisomers of 2',3'-dideoxy-2',3'-didehydrocyclopentane-adenine 5'-alpha -methylenephosphonyl-beta ,gamma -diphosphate (4) and its guanine counterpart (5), as well as several (-)-beta -L-oxathiolanenucleoside 5'-triphosphates (6-9) and (-)-beta -L-dioxolanenucleoside 5'-triphosphates (10), 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 beta  (11, 12) and endogeneous reverse transcriptases (13).

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 (4) 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 alpha , beta , and epsilon  from human placenta, reverse transcriptases from HIV and avian myeloblastosis virus, and TDT from calf thymus. We also synthesized two anomeric alpha -D-oligodeoxynucleotides and studied them as primers for TDT.


MATERIALS AND METHODS

The starting compounds, beta -L-, alpha -D- and alpha -L-2'-deoxynucleosides, were synthesized as described in Refs. 14, 15, and 16, respectively. 2'-Deoxynucleoside 5'-triphosphates were purchased from Boehringer Mannheim. alpha -d[(Tp)3T] and alpha -d[(Tp)11)T] were synthesized according to Ref. 17; beta -d[(Tp)3T] was generously provided by Dr. T. Bocharova (Institute of Molecular Genetics, Moscow, Russia).

beta -L-dNTPs, alpha -D-dNTPs, and alpha -L-dNTPs (Fig. 1) were synthesized according to Ludwig (18) 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.


Fig. 1. Stereoisomers of deoxynucleoside 5'-triphosphates.
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Table I.

Characteristics of dNTP stereoisomers

HPLC was carried out on a Daltosil C-18 column (4 µm, 150 × 4 mm) using a linear gradient of 0-25% methanol in 50 mM triethylammonium acetate (pH 6). The flow rate was 0.5 ml/min. The retention times for the corresponding beta -D-dNTPs are given in parentheses.


Compound UV spectrum, lambda max Retention time

nm min
 beta -L-dTTP 268  (pH 7) 10.5
 beta -L-dCTP 275  (pH 10) 6.5
281  (pH 2)
 alpha -D-dATP 261  (pH 7) 12.5  (12.8)
 alpha -D-dCTP 275  (pH 10) 7  (6.5)
281  (pH 2)
 alpha -D-dTTP 268  (pH 7) 11.5  (10.5)
 alpha -L-dATP 261  (pH 7) 12.5
 alpha -L-dCTP 275  (pH 10) 7
281  (pH 2)
 alpha -L-dTTP 268  (pH 7) 11.5

Enzymes and DNA

HIV reverse transcriptase was isolated according to Ref. 19. DNA polymerases alpha  and epsilon  were isolated from human placenta as described in Ref. 20; DNA polymerase beta  was purified according to Ref. 21. 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 Ref. 22. Tetradecanucleotide primers a and b (Fig. 2) and alpha -D-oligonucleotides were labeled at the 5' terminus using [gamma -32P]ATP (Radioizotop, Russia) and T4 polynucleotide kinase (Amersham Corp.) according to Ref. 23. 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 alpha ); and 10 mM Tris-HCl (pH 8.5), 5 mM MgCl2, and 1 mM dithiothreitol (for DNA polymerase beta ).


Fig. 2. Structure of the template-primers.
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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 alpha  and beta ), 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 alpha -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 (24-26), 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 beta -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 beta -L-dCTP and beta -L-dTTP as substrates for human DNA polymerases alpha , beta , and epsilon . It can be seen in Fig. 4 that beta -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 epsilon  (data not shown).


Fig. 3. Series 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) beta -L-dCTP; lane 6, as in lane 1 + 2 µM dATP + 20 µM beta -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) beta -L-dCTP.
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Fig. 4. Primer extension by DNA polymerases alpha  (A) and beta  (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) beta -L-dCTP; lanes 6, as in lanes 1 + 2 µM dATP + 20 µM beta -L-dCTP + 20 µM dGTP.
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Fig. 3B presents the results of TDT assays in the presence of beta -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 beta -L-dTTP displayed the same substrate activity as beta -L-dCTP for all DNA polymerases studied (data not shown).

It can be seen in Fig. 5 that alpha -L-dNTPs (lanes 3, 4, 6, and 7) and alpha -D-dNTPs (lanes 8, 9, 11, and 12) are not substrates for HIV reverse transcriptase. We assume that alpha -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 alpha -D-dATP completely inhibited primer extension at a [alpha -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 alpha -D-dNTPs and alpha -L-dNTPs were not utilized as substrates by human DNA polymerases (data not shown).


Fig. 5. Primer 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) alpha -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) alpha -L-dATP; lanes 8 and 9, as in lane 1 + 2 µM (lane 8) and 20 µM (lane 9) alpha -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) alpha -D-dATP.
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It is evident from Fig. 6 that two alpha -D-dTMP (lanes 10-12) and alpha -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 alpha -D-dCTP (data not shown). The alpha -L-dTTP (Fig. 6, lanes 4-6) and alpha -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.


Fig. 6. Primer 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) alpha -L-dTTP; lanes 7-9, as in lane 1 + 0.2 µM (lane 7), 2 µM (lane 8), and 20 µM (lane 9) alpha -L-dATP; lanes 10-12, as in lane 1 + 0.2 µM (lane 10), 2 µM (lane 11), and 20 µM (lane 12) alpha -D-dTTP; lanes 13-15, as in lane 1 + 0.2 µM (lane 13), 2 µM (lane 14), and 20 µM (lane 15) alpha -D-dATP.
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Then, we examined the ability of TDT to elongate oligonucleotides terminated by alpha -D-dNMP residues in the presence of different concentrations of dNTPs. It can be seen in Fig. 7 that oligodeoxynucleotides containing alpha -D-dTMP and alpha -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.


Fig. 7. Primer 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) alpha -D-dTTP; lanes 9-13, as in lane 1 + 2 µM (lane 9) and 20 µM (lanes 10-13) alpha -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.
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At the second stage of this research we evaluated alpha -d[(Tp)11T] and alpha -d[(Tp)3T] as primers for TDT in the presence of natural dNTPs and alpha -dNTP (Fig. 8, B and D). Oligonucleotides beta -d[(Tp)9T] (Fig. 8A) and beta -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.


Fig. 8. Extension of beta -d[(Tp)9T] (A), alpha -d[(Tp)11T] (B), beta -d[(Tp)3T] (C), and alpha -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.
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DISCUSSION

Our results indicate that template-dependent DNA polymerases utilize as substrates only beta -D-dNTPs, whereas for template-independent TDT alpha -D-dNTPs but not beta -L-dNTPs or alpha -L-dNTPs are substrates. It is noteworthy that alpha -D-dNTPs but not beta -L-dNTPs or alpha -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. (2) 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 beta -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 beta -L-dNTPs, many uninterpretable bands were observed. Finally, formation of oligo(dT)19 observed by Focher et al. (2) upon primer extension by DNA polymerase alpha , 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' right-arrow 5' exonuclease activity. Indeed, ion-exchange chromatography performed in Ref. 2 cannot properly separate inorganic pyrophosphate from beta -L-dTTP, because these compounds have close charges under the conditions described. Thus, it is possible that pyrophosphorolysis led to formation of beta -D-dTTP in the assay mixture and subsequent primer extension. Therefore, for our part, we additionally purified beta -L-dTTP and beta -L-dCTP by reversed-phase HPLC.

Interestingly, the affinity of 3'-modified beta -L-dNTP analogs to human DNA polymerases alpha , beta , epsilon , and gamma  and HIV reverse transcriptase drops as the bulk of the substituent is increased, O > S > CH approx  CH2 > CH2OH (Refs. 7 and 10; this paper for human DNA polymerases; Refs. 3, 7, and 9 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 beta -L-dNTP, which hinders productive dNTP binding.

It has earlier been shown (4) that carbocyclic alpha -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 alpha -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, alpha -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 (4, 27, 28). It is likely that alpha -L-dNTPs do not interact productively with TDT because of the difference in the position of the 3'-hydroxyl in L- and D-dNTPs.


Fig. 9. Structure of the TDT substrate carbocyclic alpha -D- and L-dNTP analogs I and II and the TDT inhibitor phenyl phosphonyldiphosphate III.
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Because beta -oligodeoxyribonucleotides containing one or two alpha -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 alpha -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 alpha -oligonucleotides are interesting in this respect due to their increased stability in serum (29). In this work we compared two alpha -oligothymidylates with their beta  counterparts as primers for TDT and found that all four oligonucleotides are extended by this enzyme, although the alpha -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.


FOOTNOTES

*   This work was supported by Russian Foundation for Basic Research Grants N93-04-20542 and N95-03-08142a and the French CNRS (project "Cooperation Franco-russe" 1752).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   To whom correspondence should be addressed. Tel.: 33-4-67-14-33-94; Fax: 33-4-67-04-20-29; E-mail: rayner{at}univ-montp2.fr.
1   The abbreviations used are: beta -D-dNTP, natural 2'-deoxynucleoside 5'-triphosphate; alpha -D-dNTP with N being C, T, or A, alpha -anomer at C-1' carbon of dNTP; beta -L-dNTP with N being C or T, beta -D-dNTP enantiomer; alpha -L-dNTP with N being C, T, or A, alpha -anomer at C-1' carbon of beta -L-dNTP, beta -D-dNMP, natural 2'-deoxynucleoside 5'-monophosphate; alpha -D-dNMP, alpha -anomer at C-1' carbon of dNMP, beta -L-dTMP, beta -D-dTMP enantiomer; TDT, terminal deoxynucleotidyl transferase; HIV, human immunodeficiency virus; HPLC, high pressure liquid chromatography.

ACKNOWLEDGEMENTS

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


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