(Received for publication, May 4, 1995; and in revised form, September 9, 1995)
From the
Oligonucleotides containing a specific initiation site for
polymerase -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.
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
(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.
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 -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
.
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 -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.
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 () 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.
Figure 2:
Extension from 3`-araCMP-terminated RNA
primers by pol . 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.''
Extension from the
araCMP 3` terminus using the kinetic assay requires that pol
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.
Figure 3:
Incorporation of araCMP during RNA-primed
DNA synthesis. Primase-coupled pol 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.
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 reactions were
prepared using the 40-mer (
), araCMP31 (
), araCMP23
(
), and araCMP21 (
) templates and incubated at 37
°C.
Figure 6:
Insertion of dGMP opposite araCMP from a
primer RNA by pol . 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.''
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 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.
Figure 7:
Extension from araCMP:dGMP using an RNA
primer by pol . 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.''
We used a DNA template with a specific primer initiation
sequence to study incorporation of araCMP into RNA-primed DNA fragments
by pol -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.
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 . 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.
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 . 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.
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 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.
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 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.
The primase and pol 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.