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(Received for publication, May 4, 1995; and in revised form, September 9, 1995) From the
Oligonucleotides containing a specific initiation site for
polymerase
In cells, RNA-primed DNA fragments are synthesized at origins of
replication and on lagging strands at replication
forks(1, 2) . Initiation of DNA chains requires
oligoribonucleotide synthesis to provide 3`-hydroxyl termini for DNA
polymerase elongation. The tight association between DNA polymerase
The aranucleotides
inhibit DNA replication in animal cells(3) . Inhibition by the
triphosphate forms of these analogues might result during RNA-primed
DNA synthesis by pol Oligonucleotide templates permit analysis of sequence-specific
effects of aranucleotides on RNA-primed DNA synthesis. The sequences of
some primer initiation sites have been
identified(13, 14, 15, 16) , and
these sequences support initiation in oligomer
templates(15, 16) . Using an oligonucleotide
containing an initiation site for pol
Figure 1:
The insertion of araCMP into primer
RNA. The sequence of the 40-mer DNA template and the 9-araCMP and
10-araCMP RNA products with araCMP (C* in boldface
type) 3` termini are shown (A). Primase reactions were
prepared in the absence of araCTP (B, lanes 3, 16) and in the presence of the indicated concentrations of
araCTP (B, lanes 4-14, 17, 18) as described under ``Experimental Procedures''
or with 5 pmol of recombinant primase (lanes 16-18).
Incubations were at 37 °C for 60 min. The products with araCMP 3`
termini (9-araCMP and 10-araCMP) and with CMP 3` termini (9-mer and
10-mer) are indicated. The lanes M contain the chemically
synthesized 10-mer RNA, and lane NE contains no enzyme. The 9-
and 10-Nt products were quantified, and the relative amounts of araCMP-
and CMP-containing 10-Nt (
Figure 2:
Extension from 3`-araCMP-terminated RNA
primers by pol
Extension from the
araCMP 3` terminus using the kinetic assay requires that pol
Figure 3:
Incorporation of araCMP during RNA-primed
DNA synthesis. Primase-coupled pol
Figure 4:
Primer RNA synthesis on araCMP-containing
templates. The sequence of the 40-mer template and the site of primer
synthesis is indicated. The positions of araCMP (C* in boldface type) in the templates are shown. Primase reactions
containing the indicated template were prepared and incubated at 37
°C. Lane M contains the chemically synthesized 10-mer
RNA.
Figure 5:
RNA-primed DNA synthesis on
araCMP-containing templates. Primase-coupled pol
Figure 6:
Insertion of dGMP opposite araCMP from a
primer RNA by pol
Synthesis past araCMP was measured using the RNA primer
containing an additional dGMP at the 3` terminus. The RNA 10-mer-dGMP
primer was hybridized to the araCMP21 and 40-mer templates, and
extension from the araCMP:dGMP 3` terminus by pol
Figure 7:
Extension from araCMP:dGMP using an RNA
primer by pol
We used a DNA template with a specific primer initiation
sequence to study incorporation of araCMP into RNA-primed DNA fragments
by pol The
related nucleotide analogues, FaraATP, araATP, and araCTP, might
inhibit DNA replication by different mechanisms that relate to
sequence-specific effects. Primer initiation sites are rich in
thymidine and cytosine
residues(13, 15, 16, 26) . The high
level of thymidine residues should make it more likely that FaraAMP and
araAMP might be incorporated into elongating primers by primase within
the first 6 bases, resulting in truncated primers that are not
elongated by pol During replication, RNA
primers are removed, and the resulting DNA fragments are joined by
ligase(1, 2) . It is not known which enzyme(s) removes
RNA primers, nor is it known how the RNA-DNA border is recognized. Our
results show that araCMP incorporated at the 3` terminus of an RNA
primer by primase can be elongated by pol Since araCMP is detected at internucleotide
positions(23, 24) , araCMP in the DNA template might
have an effect on initiation of RNA-primed DNA synthesis. Our results
show that araCMP positioned within the primer site reduces primer
synthesis 2-3-fold (Fig. 4). This moderate effect on
primer synthesis using araCMP-containing DNA might relate to the low
fidelity of primase(29, 30, 31) . In
contrast, araCMP positioned one nucleotide downstream from the priming
site significantly reduced primer elongation by pol The kinetic analysis reveals large differences in
elongation efficiencies of araCMP-containing RNA and DNA primers and
small differences in elongation from RNA and DNA primers using
araCMP-containing templates. The pol The primase and pol
Volume 270,
Number 44,
Issue of November 3, 1995 pp. 26664-26669
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-Primase (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-primase (pol -primase) were used to measure the
effects of cytosine arabinoside triphosphate and cytosine arabinoside
monophosphate (araCMP) in DNA on RNA-primed DNA synthesis. Primase
inserts araCMP at the 3` terminus of a full-length RNA primer with a
400-fold preference over CMP. The araCMP is elongated efficiently by
pol in the primase-coupled reaction. Extension from RNA 3`-araCMP
is 50-fold less efficient than from CMP, and extension from DNA
3`-araCMP is 1600-fold less efficient than from dCMP. Using
araCMP-containing templates, primer synthesis is reduced
2-3-fold, and RNA-primed DNA synthesis is reduced 2-8-fold.
The efficiency of polymerization past a template araCMP by pol is
reduced 180-fold during insertion of dGMP opposite araCMP and 35-fold
during extension from the araCMP:dGMP 3` terminus. These results show
that the pol -primase efficiently incorporates araCMP as the
border nucleotide between RNA and DNA and suggest that the inhibitory
effects of araC most likely result from slowed elongation of pol
and less so from inhibition of primer synthesis by primase.
(pol ) (
)and primase indicate that pol
-primase complex is responsible for RNA-primed DNA synthesis.
Primase synthesizes RNA primers that are about 10 Nts in length de
novo, and pol elongates the primers.-primase(4) . Incorporation into RNA
primers by primase and into DNA by pol is at positions opposite
complementary
nucleotides(3, 4, 5, 6, 7, 8, 9, 10) .
Primase inserts araATP and FaraATP more efficiently than
ATP(9, 11) , and pol inserts these analogues as
efficiently as deoxynucleotides(12) . Insertion of
aranucleotides terminates primer chain elongation by
primase(9, 11) , and 3`-aranucleotides in DNA decrease
elongation by pol 2000-fold (12) . In contrast,
aranucleotides at RNA 3` termini only moderately decrease elongation by
pol (9, 11) . The synthesis of nonfunctional
primers less than 7 Nts in length using homopolymer templates has made
it difficult to quantitate effects of aranucleotides during RNA-primed
DNA synthesis and to distinguish effects on primase and pol . -primase, primers with the
sequence 5`-GGAAGAAAGC-3` are generated(16) . Incorporation of
araCMP at the 3` terminus of this full-length RNA primer and its effect
on RNA-primed DNA synthesis were measured. In addition, cytosine
residues within the initiation sequence and one nucleotide downstream
were replaced with arabinocytosines. With these araCMP-containing
templates, primer RNA synthesis and DNA synthesis were measured
independently and during processive synthesis of RNA-primed DNA
fragments.
Materials
Radiolabeled nucleotides were
from Amersham. Unlabeled dNTPs, araCTP, and bovine serum albumin were
from Sigma. The NTPs were from Pharmacia Biotech Inc. Enzymes were from
Promega or U. S. Biochemical Corp. The 40-mer DNA was from Operon
Technologies. The 9-mer, 10-mer, and 11-mer DNA primers and
araCMP-containing templates (araCMP21, araCMP23, and araCMP31) were
synthesized in the Cancer Center of Wake Forest University. The 10-mer
RNA was from Oligo's Etc. pol -primase was
purified(17) , and unit definitions were as
described(17, 18) . Recombinant primase was a generous
gift of Dr. William Copeland (NIEHS, National Institutes of Health).DNA Templates and Primers
The
araCMP-containing templates were purified on urea-polyacrylamide
gels(12) , and potential gel contaminants were removed from
recovered oligonucleotides using SEP-PAK C18 cartridges (Waters). The
RNA 10-mer (5`-GGAAGAAAGC-3`) and DNA 9-mer, 10-mer, and 11-mer
(5`-GGAAGAAAG(C)(G)-3`) were gel purified, recovered, and passed
through Sephadex G-25 columns. The araCMP-terminated RNA 10-mer
(10-araCMP) was generated by incubating RNA 10-mer with T4 DNA
polymerase without dNTPs to produce a 9-mer. The 9-mer was recovered,
hybridized to the 40-mer, and elongated with Klenow (exo-) in the
presence of araCTP. The araCMP-terminated DNA 10-mer was produced by
elongating the DNA 9-mer in the presence araCTP. The RNA 10-mer-dGMP
was generated by elongation of the RNA 10-mer in the presence of dGTP.
All primers were gel purified.Primase, pol
All reactions (10 µl) contained 20 mM Tris-HCl, pH 7.5, 5 mM MgCl, and Primase-coupled pol Reactions
, 2 mM dithiothreitol, 0.1 mg/ml bovine serum albumin, and pol
-primase. Primase reactions contained 100 pmol of 40-mer template,
6.3 units of primase, and 100 µM NTPs with
[-P]ATP. The pol
reactions contained
0.25 pmol of P-5`-labeled primer hybridized to the 40-base
DNA templates at a 10:1 molar ratio (template:primer), 0.15 units of
pol
, and varying concentrations of nucleotide. Primase-coupled
pol reactions contained 100 pmol of 40-mer template, 3.8 units of
primase, 1.8 units of pol , 100 µM NTPs, and 100
µM dCTP, dGTP, TTP with 10 µM
[-P]dATP. Incubations were at 37 °C for
the indicated times, and reactions were stopped by addition of EDTA to
5 mM. Primase and pol
reactions were subjected to
electrophoresis through 20% urea-polyacrylamide gels. Dried gels were
quantified using AMBIS radioanalytic imaging system (San Diego) or
phosphorimagery (Molecular Dynamics). Primase-coupled pol
reactions were processed for scintillation spectroscopy by collecting
acid-insoluble products on glass fiber disks.
Insertion of araCMP into Full-length Primer
RNA
pol -primase generates RNA-primed DNA fragments by
synthesizing RNA primers greater than seven nucleotides in length and
polymerizing DNA onto the primers. An oligonucleotide containing an
initiation site for primer synthesis (16) was used to measure
araCMP insertion opposite guanosine at the 3` terminus of a full-length
primer (Fig. 1A). In the absence (Fig. 1B, lane 3) and in the presence of
increased concentrations of araCTP (Fig. 1B, lanes
4-14), primase generated RNA primers 9-11 Nts in
length. This result demonstrates that pol -primase synthesizes
full-length primer RNA in the presence of araCTP using this template.
Insertion of araCMP was apparent from the appearance of the 9-araCMP
and 10-araCMP oligomers that migrate more rapidly in the gel than those
generated in the absence of araCTP. The araCMP-containing primers were
detected even in the presence of a 1000-fold excess of the
complementary substrate, CTP (Fig. 1B, lane
4). The efficiency of araCMP insertion into primer RNA was
determined by quantifying the 9- and 10-base products containing CMP
and those of altered mobility containing araCMP. A linear relationship
was demonstrated between the fraction of primers containing CMP at the
3` terminus and the [CTP]/[araCTP] (Fig. 1C). The inverse slope of this line is equal to
the relative (k/K
)/(k
/K
)
and is an estimate of the relative efficiencies of araCTP and CTP as
substrates for primase(19) . Quantitation indicates that araCTP
is a 400-fold better substrate for primase than is CTP. To confirm that
primase, not pol
, inserts araCMP at this site, primase reactions
devoid of pol were performed using recombinant primase (Fig. 1B, lanes 16-18). Generation of
the araCMP-terminated 9- and 10-base products demonstrates that primase
is responsible for araCMP insertion.
) and 9-Nt () primers were
determined for each araCTP concentration (C). The sequence of
the product band that migrates at the 11-mer position has not been
determined.
Extension from the araCMP-terminated RNA
Primer
Extension from an araCMP-terminated RNA primer could
lead to incorporation at internucleotide positions. A steady state
kinetic assay (20, 21) was used to measure the
efficiency of extension from the araCMP primer RNA by pol . The
RNA 10-araCMP and 10-mer primers were hybridized to the 40-mer template
and extended with DNA pol (Fig. 2). In the presence of
increased concentrations of the next complementary nucleotide,
increased amounts of 11-mer were detected. Extension from both primers
demonstrates saturation kinetics, and the apparent K and V
values for the next nucleotide
addition were determined (Table 1). The V/K values indicate that
pol extends the araCMP-terminated RNA primer 50-fold less
efficiently than the CMP-terminated primer. This result contrasts the
2000-fold decrease in extension efficiency that is observed from an
araCMP-terminated DNA primer ( Table 1and (12) ). These
results show that pol extends araCMP-terminated RNA primers more
efficiently than araCMP-terminated DNA primers.
. The 10-araCMP and 10-mer RNA primers were
hybridized to the 40-mer DNA template to generate dGMP:araCMP and
dGMP:CMP 3` termini. The template:primers were extended (90 s, 37
°C) in pol reactions containing the indicated concentrations
of dGTP. Oligonucleotide products from the araCMP () and CMP
() 3` termini are shown top, left, and right, respectively. Quantitation was as described under
``Experimental Procedures.''
first bind to the template:primer then catalyze the addition of
deoxynucleoside monophosphate. However, pol -primase synthesizes
an RNA primer and then elongates the primer without dissociating from
the DNA template(22) . Therefore, it was of interest to
determine if insertion of araCMP at the 3` terminus of a full-length
RNA primer affects elongation by pol in a processive reaction.
Primase-coupled pol reactions were performed, and the rate of
RNA-primed DNA synthesis in the presence of araCTP (6 fmol of dAMP/min)
was equal to the rate in the presence of CTP (7 fmol of dAMP/min). To
confirm that araCMP is incorporated as the border nucleotide,
primase-coupled pol reactions were performed, and araCMP in the
product was verified by its migration on a polyacrylamide gel (Fig. 3). To limit DNA synthesis by pol and to eliminate
the possibility that dCTP might be inserted as the first
deoxynucleotide, only dGTP and ddATP were included for elongation in
these reactions. The product containing araCMP (11-araCMP) migrates
more slowly in the gel than the 11-mer containing CMP (Fig. 3,
compare lanes 4 and 5). In the absence of CTP,
primase synthesized a 9-mer, and no misincorporation of NTP was
detected (Fig. 3, lane 3). These results indicate that
araCMP is efficiently incorporated as the border nucleotide between RNA
and DNA during RNA-primed DNA synthesis by pol -primase.
reactions were prepared as
described under ``Experimental Procedures'' containing 100
µM dGTP, ddATP, [-P]ATP, GTP,
UTP, and araCTP (lane 4) or CTP (lane 5). Primase
reactions contained 100 µM
[
-P]ATP, GTP, UTP (lane 3), and
araCTP (lane 1) or CTP (lane 2). Incubation was at 37
°C for 60 min. The positions of migration of the products are
indicated.
Primer RNA Synthesis on araCMP-containing
Templates
The DNA isolated from araC-treated cells contains
araCMP at internucleotide positions(23, 24) . To test
the effects of araCMP in DNA on RNA-primed DNA synthesis by pol
-primase, three araCMP-containing templates were prepared (Fig. 4). For two of the templates, a dCMP within the priming
site (Nt 31 or Nt 23) was replaced with araCMP. For the third template,
the dCMP positioned at the first nucleotide downstream from the priming
site (Nt 21) was changed to araCMP. Primase initiates synthesis by
insertion of the first 5`-GTP at nucleotide 31. Nucleotide 23 is seven
nucleotides from the initiation start site(16) . These two
positions were selected to test for differences in primer synthesis
that might relate to the position of araCMP within the priming site.
Nucleotide 21 was selected to measure the effect of araCMP in the
template at the primase to pol switch position. Synthesis of RNA
primers using the araCMP-containing templates was determined in time
course reactions (Fig. 4). Products 9-11 Nts in length are
detected using all three of the araCMP-containing templates.
Quantitation of the oligomer products shows that the rates of primer
synthesis are reduced 2-fold using araCMP31 (Fig. 4, lanes
7-11) and araCMP23 (Fig. 4, lanes
12-16) and 3-fold using araCMP21 (Fig. 4, lanes
17-21) relative to the 40-mer template (Fig. 4, lanes 2-6). These results indicate that primase
initiates primer synthesis and generates full-length RNA primers at
priming sites containing araCMP at a reduced rate.
Primase-coupled pol
The rates of RNA-primed DNA
synthesis using the araCMP-containing templates were reduced relative
to the 40-mer template (Fig. 5). The levels of inhibition were
dependent upon the positions of araCMP within the DNA templates. With
the araCMP31 and araCMP23 templates, primase-coupled pol Synthesis Using
araCMP-containing Templates
synthesis was reduced 2- and 3-fold, respectively. The araCMP
positioned at the presumed site of primase to pol switching on
the araCMP21 template had the greatest inhibitory effect, reducing
primase-coupled pol synthesis by 8-fold (Fig. 5). These
results likely reflect, in part, the reduced rates of primer synthesis
on these templates, while the araCMP positioned at the primase to
polymerase switch further inhibits elongation by pol .
reactions were
prepared using the 40-mer (), araCMP31 (), araCMP23
(
), and araCMP21 (
) templates and incubated at 37
°C.
Synthesis Past araCMP from an RNA Primer by pol
To quantitate the effect of araCMP positioned in the
DNA template at the RNA-DNA border on elongation by pol , the
kinetic assay was used to measure DNA synthesis independently from RNA
primer synthesis (Fig. 6). Synthesis past araCMP requires
insertion of dGMP opposite araCMP and extension from the
3`-araCMP:dGMP. In separate reactions, the 10-mer RNA primer was
hybridized to the araCMP21 and 40-mer templates and incubated with pol
and increased concentrations of dGTP. Quantitation of oligomer
products (Table 2) shows that the K value
for addition of dGMP opposite araCMP is 100 µM. The K value for addition of dGMP opposite dCMP is 0.11
µM. The V values are more similar
with a 5-fold greater V value obtained using the
araCMP21 template. The relative V/K values for these
reactions indicate that pol inserts dGMP opposite araCMP 180-fold
less efficiently than it inserts dGMP opposite dCMP during RNA primer
elongation.
. The 10-mer RNA primer was hybridized to the
araCMP21 () and 40-mer () templates and extended (60 s, 37
°C) in pol
reactions containing the indicated concentrations
of dGTP. Oligonucleotide products were quantified as described under
``Experimental Procedures.''
was measured (Fig. 7). The K values for next nucleotide
addition are 2.4 µM using the araCMP21 and 0.072
µM using the 40-mer templates (Table 2). The
relative V/K values for
extension from the araCMP:dGMP and dCMP:dGMP termini indicate that
extension from araCMP:dGMP by pol is 35-fold less efficient than
from dCMP:dGMP. These results suggest that araCMP positioned in the
template as the border nucleotide between RNA primer and DNA synthesis
has a greater inhibitory effect on RNA-primed DNA synthesis than does
araCMP positioned in the template within the RNA priming site.
. The 10-mer-dGMP primer was hybridized to the
araCMP21 () and the 40-mer () templates and extended (60
s, 37 °C) in pol
reactions containing the indicated
concentrations of dATP. Oligonucleotide products were quantified as
described under ``Experimental
Procedures.''
Synthesis Past araCMP from a DNA Primer by pol
To test the relative effects of araCMP in DNA on pol
polymerization from RNA versus DNA, a kinetic analysis
of araCMP bypass using DNA primers was performed (Table 2). The
position for DNA primer elongation was identical to that chosen for RNA
primer elongation. A DNA 10-mer was hybridized to the araCMP21 and
40-mer templates and elongated with pol in the presence of dGTP.
Similarly, a DNA 11-mer was hybridized to the two templates and
elongated with pol . Quantitation indicates that insertion of dGMP
opposite araCMP is 50-fold less efficient than insertion opposite dCMP.
Extension from the araCMP:dGMP terminus is 160-fold less efficient than
from the dCMP:dGMP terminus. These results are similar to those
obtained using RNA primers and suggest that pol synthesis past
araCMP is not influenced by the nature of the primer strand. However,
the rate of elongation past araCMP in DNA from both RNA and DNA primers
is at least 10-fold more efficient than elongation from an araCMP 3`
terminus ( Table 1and Table 2), indicating that the
position of araCMP in the nascent strand reduces the rate of elongation
to a greater extent than does araCMP in the template strand.
-primase. Our results demonstrate that primase incorporates
araCMP opposite dGMP as the 3`-terminal nucleotide of a full-length RNA
primer very efficiently and that pol elongates the
araCMP-terminated RNA primer. These data suggest that araCMP might be
incorporated efficiently into RNA-primed DNA fragments at certain
positions during discontinuous DNA synthesis in cells. Previous in
vitro studies using homopolymer and bacteriophage templates have
shown that aranucleotides are potent inhibitors of RNA-primed DNA
synthesis by pol
-primase(3, 4, 5, 6, 7, 8, 9) .
Using poly(dT) templates, aranucleotides are detected in truncated RNA
primers exclusively at 3` termini, suggesting that primer chain
termination is a likely mechanism of primase
inhibition(9, 11) . The truncated primers 2-6
Nts are not sufficient in length to support subsequent DNA synthesis by
pol (25) . However, primers of at least 7 Nts in length
containing a 3`-terminal aranucleotide are elongated by DNA pol
(Refs. 7, 9, 11, and this study). Since aranucleotides are incorporated
into RNA primers at positions of correct base pairing, it is apparent
that the DNA template sequence is an important consideration.. In contrast, the less frequent guanosines in
initiation sites make it less likely that nonfunctional primers are
generated in the presence of araCTP. Previous results using cell
lysates indicate that FaraATP inhibits and araCTP stimulates primer RNA
synthesis(8) . These results might be explained by the
generation of a high level of nonfunctional primers in the presence of
FaraATP, resulting in the inability to initiate DNA synthesis. In
contrast, araCMP might be incorporated more frequently into full-length
RNA primers. Our results show that DNA pol elongates an
araCMP-terminated RNA primer more efficiently than an araCMP-terminated
DNA primer. Kinetic measurements of extension efficiencies from araCMP
termini show a 50-fold reduction for RNA and a 2000-fold reduction for
DNA ( Table 1and (12) ). The failure to elongate
araCMP-containing DNA 3` termini is also apparent in the
chain-termination effects associated with
araCTP(10, 23, 27) . Furthermore, extension
from araCMP-terminated RNA primers appears to be efficient during
primase-coupled pol synthesis (Fig. 1C). Thus, a
greater level of RNA primer synthesis in the presence of araCTP might
be explained by shorter RNA-primed DNA fragments resulting from
inhibition of DNA chain elongation and subsequent reinitiation at
regions of single-stranded DNA downstream.. The rate of RNA primer
removal in the presence of araCTP is reduced(28) . It is
possible that araCMP at the RNA-DNA border is not removed, resulting in
5`-araCMP in DNA fragments. It has been shown that DNA fragments with a
3`-araCMP and 5`-deoxynucleoside monophosphate can be
ligated(27) . Ligation of 5`-araCMP and 3`-deoxynucleoside
monophosphate has not been tested. The inefficient removal of araCMP
from the 3` terminus of an RNA primer and subsequent ligation of DNA
fragments might lead to incorporation of araCMP at internucleotide
positions. in a kinetic
analysis and in the primase-coupled pol reaction ( Table 2and Fig. 5). These results are similar to those of
Mikita and Beardsley(27) , who showed that araCMP in the DNA
template slowed replication bypass for some polymerases. Thus, the
major effect of araCMP in the DNA template appears to be on DNA
polymerization. discriminates against araCMP
insertion into DNA 4-fold better using RNA primers than DNA primers (7) . Similarly, insertion of dGMP opposite araCMP from an RNA
primer is 4-fold less efficient than from a DNA primer (Table 2).
However, extension from the RNA 10-mer-dGMP positioned opposite araCMP
was 5-fold more efficient than from the DNA 11-mer. Overall, the
kinetic analysis shows that the efficiency of bypass of araCMP in the
template by pol -primase is very similar for RNA and DNA primers. are very likely target enzymes for
aranucleotides during genomic DNA replication. Using an oligonucleotide
template, we have measured the multiple effects that araC has on the
synthesis of RNA-primed DNA fragments. Most significantly, we show that
araCMP is efficiently incorporated as the border nucleotide between RNA
and DNA by the pol -primase complex.
), DNA polymerase ; araCMP,
cytosine arabinoside monophosphate; araC,
1-
-D-arabinofuranosylcytosine; araA,
9--D-arabinofuranosyladenosine; FaraATP,
9--Darabinofuranosyl-2-fluoroadenine 5`-triphosphate;
NTP, nucleoside triphosphate; Nt(s), nucleotide(s).
We thank Eric Roesch (WFU Cancer Center) for synthesis
of oligonucleotides and Dr. William Copeland (NIEHS, National
Institutes of Health) for recombinant primase.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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