(Received for publication, November 5, 1996)
From the 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
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
,
, and
from human placenta and
reverse transcriptases of human immunodeficiency virus and avian
myeloblastosis virus incorporate into the DNA chain only natural
-D-dNTPs, whereas calf thymus terminal
deoxynucleotidyl transferase incorporates two nucleotide residues of
-D-dNTP and extends the resulting oligonucleotide in the
presence of
-D-dNTPs. The latter enzyme also extended
-anomeric D-oligodeoxynucleotide primers in the presence
of
-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.
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
-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 -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
[
-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
-L-dTTP on mammalian mitochondrial
DNA polymerase
was weaker than that observed for HIV reverse
transcriptase. DNA polymerases
and
did not utilize
-L-dTTP as a substrate (1). Focher et al. (2)
have studied the substrate properties of
-L-dTTP toward
human DNA polymerases
,
,
,
, and
, 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
,
,
, and
did not
incorporate nucleotide residues of this compound into the DNA chain,
whereas the other enzymes extended the primer by one or more
-L-dTMP residues. Specifically, DNA polymerases
and
Klenow fragment incorporated two
-L-dTMP residues, and HIV reverse transcriptase elongated the primer by up to 3 or 4
-L-dTMP residues.
Furthermore, it has been shown (3) that
2,3
-dideoxy-
-L-thymidine 5
-triphosphate and
2
,3
-dideoxy-2
,3
-didehydro-
-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
-D-isomers. The
-L-stereoisomers of
2
,3
-dideoxy-2
,3
-didehydrocyclopentane-adenine 5
-
-methylenephosphonyl-
,
-diphosphate (4) and its guanine counterpart (5), as well as several
(
)-
-L-oxathiolanenucleoside 5
-triphosphates (6-9)
and (
)-
-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 (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
,
, and
from human placenta, reverse transcriptases from HIV and avian
myeloblastosis virus, and TDT from calf thymus. We also synthesized two
anomeric
-D-oligodeoxynucleotides and studied them
as primers for TDT.
The starting compounds, -L-,
-D-
and
-L-2
-deoxynucleosides, were synthesized as
described in Refs. 14, 15, and 16, respectively. 2
-Deoxynucleoside
5
-triphosphates were purchased from Boehringer Mannheim.
-d[(Tp)3T] and
-d[(Tp)11)T] were
synthesized according to Ref. 17;
-d[(Tp)3T] was
generously provided by Dr. T. Bocharova (Institute of Molecular
Genetics, Moscow, Russia).
-L-dNTPs,
-D-dNTPs, and
-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.
|
HIV reverse transcriptase was isolated
according to Ref. 19. DNA polymerases and
were isolated from
human placenta as described in Ref. 20; DNA polymerase
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 -D-oligonucleotides were
labeled at the 5
terminus using [
-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
); and 10 mM Tris-HCl (pH 8.5), 5 mM MgCl2,
and 1 mM dithiothreitol (for DNA polymerase
).
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
and
), 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
-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.
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 -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
-L-dCTP and
-L-dTTP as substrates for human DNA polymerases
,
, and
. It can be seen in Fig. 4 that
-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
(data not
shown).
Fig. 3B presents the results of TDT assays in the presence
of -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
-L-dTTP displayed the same substrate activity as
-L-dCTP for all DNA polymerases studied (data not
shown).
It can be seen in Fig. 5 that -L-dNTPs
(lanes 3, 4, 6, and 7) and
-D-dNTPs (lanes 8, 9,
11, and 12) are not substrates for HIV reverse
transcriptase. We assume that
-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
-D-dATP completely inhibited primer extension at a
[
-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
-D-dNTPs and
-L-dNTPs were not utilized
as substrates by human DNA polymerases (data not shown).
It is evident from Fig. 6 that two
-D-dTMP (lanes 10-12) and
-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
-D-dCTP (data not shown). The
-L-dTTP
(Fig. 6, lanes 4-6) and
-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.
Then, we examined the ability of TDT to elongate oligonucleotides
terminated by -D-dNMP residues in the presence of
different concentrations of dNTPs. It can be seen in Fig.
7 that oligodeoxynucleotides containing
-D-dTMP and
-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.
At the second stage of this research we evaluated
-d[(Tp)11T] and
-d[(Tp)3T] as primers for TDT
in the presence of natural dNTPs and
-dNTP (Fig. 8,
B and D). Oligonucleotides
-d[(Tp)9T] (Fig. 8A) and
-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.
Our results indicate that template-dependent DNA
polymerases utilize as substrates only -D-dNTPs,
whereas for template-independent TDT
-D-dNTPs but not
-L-dNTPs or
-L-dNTPs are substrates. It is noteworthy that
-D-dNTPs but not
-L-dNTPs or
-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 -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
-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
, 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
Ref. 2 cannot properly separate inorganic pyrophosphate from
-L-dTTP, because these compounds have close charges
under the conditions described. Thus, it is possible that
pyrophosphorolysis led to formation of
-D-dTTP in the
assay mixture and subsequent primer extension. Therefore, for our part,
we additionally purified
-L-dTTP and
-L-dCTP by reversed-phase HPLC.
Interestingly, the affinity of 3-modified
-L-dNTP
analogs to human DNA polymerases
,
,
, and
and HIV reverse
transcriptase drops as the bulk of the substituent is increased, O > S > CH
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
-L-dNTP, which hinders productive dNTP binding.
It has earlier been shown (4) that carbocyclic -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
-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,
-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
-L-dNTPs do not interact
productively with TDT because of the difference in the position of the
3
-hydroxyl in L- and D-dNTPs.
Because -oligodeoxyribonucleotides containing one or two
-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
-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
-oligonucleotides are interesting
in this respect due to their increased stability in serum (29). In this
work we compared two
-oligothymidylates with their
counterparts
as primers for TDT and found that all four oligonucleotides are
extended by this enzyme, although the
-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.
We are grateful to Dr. L. Victorova
(Engelhardt Institute of Molecular Biology, Moscow, Russia) for
providing DNA polymerase and to Drs S. Spadari and F. Focher
(Instituto di Genetica Biochimica ed Evoluzionistica, Pavia, Italy) for
useful discussion.