(Received for publication, April 3, 1997)
From the Department of Biochemistry, Center in Molecular Toxicology and The Vanderbilt Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
Propanodeoxyguanosine (PdG) is a model for
several unstable exocyclic adducts formed by reaction of DNA with
bifunctional carbonyl compounds generated by lipid peroxidation. The
effect of PdG on DNA synthesis by human DNA polymerase was
evaluated using template-primers containing PdG at defined sites. DNA
synthesis was conducted in vitro and the products were
analyzed by polyacrylamide gel electrophoresis and DNA sequencing. The
extent of PdG bypass was low and the products comprised a mixture of
base pair substitutions and deletions. Sequence analysis of all of the
products indicated that the deoxynucleoside monophosphate incorporated
"opposite" PdG was complementary to the base 5
to PdG in the
template strand. These findings are very similar to recent results of
Efrati et al. (Efrati, E., Tocco, G., Eritja, R., Wilson,
S. H., and Goodman, M. F. (1997) J. Biol.
Chem. 272, 2559-2569) obtained in DNA replication of
template-primers containing abasic sites and suggest that PdG is a
non-informational lesion when acted upon by polymerase (pol)
. In
addition to base pair substitutions and one- or two-base deletions, a
four-base deletion was observed and the mechanism of its formation was
probed by site-specific mutagenesis. The results indicated that this
deletion occurred by one-base insertion followed by slippage to form a
four-base loop followed by extension. All of the observations on pol
replication of PdG-containing template-primers are consistent with
a mechanism of lesion bypass that involves template slippage and dNTP
stabilization followed by deoxynucleoside monophosphate incorporation
and extension. This mechanism of PdG bypass is completely different
than that previously determined for the Klenow fragment of DNA
polymerase I and is consistent with recent structural models for DNA
synthesis by pol
.
Mutations are the cause of genetic disease and arise during the copying of normal or damaged DNA templates. The factors that determine the frequency and types of mutations include the identity of the DNA polymerase, the composition of the deoxynucleotide pool, the local sequence context of the template, and, in the case of damaged templates, the structure of the DNA lesion (1-16). Our laboratory and others have investigated the effect of propanodeoxyguanosine (PdG)1 on the fidelity of DNA replication in vivo and in vitro (17-22). PdG has been used as a model with which to assess the biological effects of several unstable DNA adducts derived from the reaction of dG residues with bifunctional aldehydes (23, 24). PdG is a relatively small lesion that completely blocks Watson-Crick base pairing but induces little distortion in the local structure of DNA molecules to which it is introduced (25, 26). Transformation of recombinant viral genomes or shuttle vectors containing site-specifically positioned PdG residues into bacterial and mammalian cells demonstrates that PdG is highly mutagenic and capable of inducing a range of base pair substitutions and frameshift mutations in vivo (17-19, 22). Likewise, PdG induces frameshift and base pair substitution mutations during in vitro replication of adducted template-primers (20, 21).
The outcome of replication of DNA molecules containing PdG is highly
sequence context dependent (20). Using the Klenow fragment (exo+ or exo) of DNA
polymerase I, we have demonstrated that the identity of the base pair
3
to PdG in the template strand influences the choice of dNMP
incorporated opposite PdG (20). More important, is the identity of the
base 5
to PdG in the template strand, which determines the outcome of
synthesis beyond PdG (20). When the 5
base can hydrogen bond to the
base opposite PdG at the primer terminus, slippage and extension occur
to generate one-base deletions at the site of the PdG residue (20, 21).
When the 5
base is T and the primer terminus contains A, the Klenow
fragment extends the primer to a full-length product (20). The
inability of the Klenow fragment to extend template-primers that
contain C residues 5
to PdG to full-length products correlates to the results of in vivo mutagenesis experiments in which PdG
induces transitions to A and transversions to T when the base 5
to it is T but not when it is C (18, 27).
To explore the generality of these observations on the replication of
PdG by other DNA polymerases, we investigated the replication of
PdG-containing template-primers by DNA polymerase (pol
). Pol
is a small protein (39 kDa) that lacks a proofreading exonuclease and is highly error prone (28, 29). It plays a role in gap-filling during mammalian DNA repair that is analogous to the role of DNA polymerase I in Escherichia coli (30-34). However, recent
results suggest that pol
also may play a role in mutagenic bypass
of lesions that block replication (e.g. cis-Pt adducts and
abasic sites) (35-37). Furthermore, a high resolution crystal
structure of pol
is available which provides a structural framework
with which to interpret the results of in vitro replication
experiments (38, 39). The preliminary results of our studies on the
replication by pol
of template-primers containing PdG residues
appeared qualitatively similar to the results obtained with the Klenow fragment. However, more detailed analysis revealed that the factors that control deoxynucleoside-monophosphate insertion opposite PdG and
extension to full-length are completely different. The structure of pol
suggests a possible explanation for these events that lead to PdG
bypass and mutation.
Deoxynucleoside 5-triphosphates
(ultrapure) were purchased from Pharmacia Biotech Inc. (Piscataway,
NJ). [
-32P]ATP (3000 Ci/mmol) and the Maxam-Gilbert
Sequencing System were from NEN Life Sciences Products (Boston, MA).
Osmium tetroxide (4 weight % solution in water) was purchased from
Aldrich Chemical Co.
1,N2-(1,3-Propano)-5
-O-DMT-3
-O-[2-cyanoethoxy-N,N-diisopropyl
phosphino]-2
-deoxyguanosine was obtained from Chem-Master
International Inc. (Stony Brook, NY). T4 polynucleotide
kinase and DNA polymerase I Klenow fragment (exo+) were
obtained from Boehringer Mannheim. Human DNA polymerase
was
purchased from Chimerx (Madison, WI).
The 5-dimethoxytrityl-protected
phosphoramidites were incorporated into oligonucleotides by Midland
Certified Reagent Co. (Midland, TX). The following 19-mer
sequences (X = PdG) were synthesized: 19-mer 5
-A-PdG-C-3
sequence, 5
-TATCGCGAXCGGCATGAGC-3
(X = PdG); 19-mer
5
-C-PdG-C-3
sequence, 5
-TATCGCGCXCGGCATGAGC-3
; 19-mer 5
-G-PdG-C-3
sequence, 5
-TATCGCGGXCGGCATGAGC-3
; 19-mer 5
-T-PdG-C-3
sequence,
5
-TATCGCGTXCGGCATGAGC-3
and 19-mer 5-dG
5-dA 5
-G-PdG-C-3
sequence, 5
-TATCACGGXCGGCATGAGC-3
. The primer sequence was as
follows: 5
-GCTCATGCCG-3
. The primer and all the unmodified
19-mer templates were obtained from the Molecular Genetics Core
Laboratory of the Vanderbilt Center in Molecular Toxicology.
Oligonucleotide sizing markers (8-32 bases) were obtained from
Pharmacia Biotech Inc.
The template oligonucleotides were purified by the vendor by anion
exchange high performance liquid chromatography. The 10-mer primer and
the 19-mer unmodified templates were obtained in the 5-dimethoxytrityl-protected form and were purified by nucleic acid
purification columns (NEN Life Science Products). All the adducted and
unadducted templates, and the primer were subjected to routine PAGE
purification before use. The final purities of all the oligonucleotides
were estimated by PAGE to be in excess of 98%.
5-End-labeling
of the primers (2 µM) was carried out using
T4 polynucleotide kinase and the labeled primers were
purified by bio-spin columns (Bio-Rad). Annealing of template-primers
was carried out by mixing template and 32P-labeled primer
in a molar ratio of 5:1 and incubating for 5 min at 90 °C followed
by slow cooling to room temperature in an annealing buffer comprising
50 mM Tris-HCl (pH 7.4), 50 µg/ml bovine serum albumin,
and 5 mM MgCl2. The template-primer mixture (100 nM primer) was incubated in 10 µl of buffer
containing 50 mM Tris-HCl (pH 6.5), 4 mM
-mercaptoethanol, 8 mM MgCl2, 4 mM dithiothreitol, and 2 mg/ml bovine serum albumin with
0.1 unit of Klenow fragment (exo+) or 4 units of pol
in
the presence of normal dNTPs (100 µM). Incubations were
carried out for 30 min at 25 °C and quenched by adding 10 µl of 10 mM EDTA in 90% formamide. The reaction products were
analyzed by electrophoresis (20% PAGE) using the ultrapure Sequagel
sequencing system (National Diagnostics, Atlanta GA). The positions of
the bands were established by autoradiography and PhosphorImager
analysis (Molecular Dynamics, Sunnyvale CA). To determine the sequences
of the primer extension products, reactions were carried out on a
10-20-fold higher scale and the products were separated by PAGE. The
bands were cut out of the gel and extracted by shaking overnight in
distilled water. The extracted products were purified by ethanol
precipitation and ultracentrifugation then subjected to Maxam-Gilbert
chemical sequencing and a T-specific reaction (40, 41).
The effect of PdG on
DNA replication catalyzed by pol was determined with
site-specifically modified template-primers (19-mer:10-mer) containing
PdG at the eleventh position from the 3
end of the template. A set of
four PdG-adducted templates were utilized in which the base 5
to the
lesion was A, C, G, or T. The adducted templates were annealed to
32P-labeled 10-mer primers and the template-primers were
used in primer extension assays carried out in the presence of all four dNTP's. The products of extension were analyzed by separation on a
20% denaturing polyacrylamide gel and detected with a
PhosphorImager. When DNA synthesis was conducted on non-adducted
template-primers, each of the primers was extended completely to a
full-length 19-mer product (data not shown). However, when PdG was
present in the template, a significant block to replication was
observed in each template-primer at the position of the adduct (Fig.
1A). Bypass of the adduct was
observed to the extent of 2-3% of that observed with unmodified
template-primers. The extended primers comigrated with 19-mer standards
when the base 5
to PdG in the template was A, G, or T and with 18-mer
standards in all sequence contexts. In addition, a 17-mer product was
observed when C was 5
to PdG and a 15-mer product was observed when G
was 5
to PdG. Parallel experiments were conducted with the Klenow
fragment (exo+) of DNA polymerase I for comparison (Fig.
1B). The overall product pattern resembled that generated by
pol
except that the 15-mer product detected in the 5
-G-PdG-C
sequence context was absent in reactions catalyzed by the Klenow
fragment.
Deoxynucleotide Incorporation Opposite PdG
Previous work from
our laboratory has shown that the Klenow fragment incorporates dGMP and
dAMP residues opposite PdG (dGMP > dAMP) but preferentially
extends the template-primer containing PdG:dAMP at the primer terminus
(20). The similarity in the profile of extension products generated by
the Klenow fragment and pol (Fig. 1) implied that a similar
mechanism might describe the course of adduct bypass by pol
. To
test this, we performed single deoxynucleotide incorporation
experiments with each of the template-primers. Contrary to our
expectations, a constant ratio of incorporation of dGMP:dAMP was not
observed but the identity of the dNMP incorporated opposite PdG changed
with each sequence context. The principal dNMP incorporated in each
case was the one complementary to the base 5
to PdG on the template
strand (Fig. 2).
Sequence Analysis of the Bypass Products
The dependence of
the identity of the dNMP incorporated opposite PdG on the nature of the
base 5 to PdG prompted us to determine the sequence of all the
extension products formed from each template-primer. The full-length
(19-mer) and base-deleted (15-, 17-, and 18-mer) products were eluted
from the polyacrylamide gels and subjected to Maxam-Gilbert chemical
sequencing with T-specific reactions where required. In cases where
multiple bands were observed opposite the adduct site in a sequence
analysis gel, the makeup of the major products was established by
comparing the intensity of the bands at the site of the adduct with
those at the neighboring bases in each lane. A dramatic difference was
observed in the specificity of base fixation opposite the adduct in the
full-length products synthesized by pol
and the Klenow fragment.
Fig. 3 displays the PhosphorImages of the
sequencing gels of full-length products synthesized by pol
on
5
-A-PdG-C-3
, 5
-G-PdG-C-3
, and 5
-T-PdG-C-3
template sequences. The
major full-length product in each of the sequence contexts resulted
from fixation of the dNMP complementary to the base 5
to PdG. In the
case of the 5
-A-PdG-C-3
sequence context, the band in the T lane was
the most prominent, indicating that the major full-length product arose
by incorporation of dTMP opposite PdG (Fig. 3A). In the
5
-G-PdG-C-3
sequence context, sequencing of the full-length product
shown in Fig. 3B, revealed predominant insertion of dCMP
across from PdG, whereas in the sequence context in which T was the 5
to PdG, the full-length product derived by the incorporation of dAMP
opposite the adduct (Fig. 3C). A small fraction of
full-length products in 5
-A-PdG-C-3
and 5
-G-PdG-C-3
sequences
originated by incorporation of dAMP across from the adduct.
In contrast to the diverse array of deoxynucleotides incorporated
opposite PdG into full-length bypass products by pol , the Klenow
fragment displayed a consistent specificity of dNMP fixation opposite
PdG regardless of the sequence context giving rise to the full-length
products. Sequence analysis of the 19-mer products obtained with the
Klenow fragment revealed that they originated by exclusive
incorporation of dAMP opposite PdG in each of the three sequences (Fig.
4). Because the bands in the T + C
lane of Fig. 4A were not very clear, a separate
T-specific reaction was carried out for the 5
-A-PdG-C-3
sequence and
the analysis showed no band at the site of the adduct, which confirmed the exclusive insertion of dAMP opposite the adduct (data not shown)
(41).
Sequence analysis of the 18-mer bands synthesized by pol on each of
the four adducted template-primers is presented in Fig. 5. In each case, the products were the
result of a one-base deletion (C) at the adduct site. Furthermore,
deletion of CpG was observed on analysis of the band running parallel
to a 17-mer that was synthesized from the 5
-C-PdG-C-3
sequence
context (Fig. 6).
Mechanism of Formation of the Four-base Deleted Product by Pol
Extension of the 5-G-PdG-C-3
template-primer by pol
produced a 15-mer product that was not formed by the Klenow fragment (Fig. 1). Sequence analysis of the eluted product indicated that it
arose by the deletion of the four-base sequence PdG-CGG (Fig. 7). This deletion could have occurred by
two routes, one involving four-base slippage prior to incorporation of
dNMP into the primer (Fig. 8, Model
I) and the other involving polymerase-mediated incorporation of
dCMP followed by slippage to form a four-base loop (Fig. 8, Model
II). To elucidate the pathway of the four-base deletion product,
primer extension was conducted with a PdG-adducted template in which
the G residue four bases 5
to PdG was changed to A (5-G
5-A
template). If slippage occurred prior to incorporation of a dNMP,
changing the identity of the fourth base 5
to PdG should have no
effect because the altered residue is not part of the slippage
intermediate. On the other hand, if dCMP was incorporated opposite PdG
before slippage, changing the identity of the base four residues 5
to
PdG should significantly inhibit deletion because of the loss of the
terminal base pair in the subsequent slippage intermediate (Fig.
9). In fact, replication of the
5-G
5-A-substituted template-primer by pol
did not yield any band
with an electrophoretic mobility of a 15-mer (Fig.
10).
Our initial comparative studies of replication of PdG-containing
template-primers by pol and the Klenow fragment suggested that the
two enzymes act in a qualitatively similar fashion. Both enzymes are
capable of bypassing the PdG residue albeit with low efficiency and
both enzymes extend the primer to full-length when the base 5
to PdG
is G, A, or T. However, analysis of the dNMP inserted opposite PdG
revealed dramatic differences between pol
and the Klenow fragment.
Whereas the Klenow fragment incorporates G and A residues opposite PdG
in a roughly constant ratio regardless of the sequence context (20),
pol
incorporated residues complementary to the base 5
to PdG in
the template strand. Changing the identity of the 5
base changes the
identity of the inserted base. These findings indicate that the Klenow
fragment uses PdG as a template base whereas pol
uses the base 5
to PdG as the template (Fig. 11).
The ability of pol to use the 5
base as a template suggests that
it can stabilize a template-primer structure that is formally equivalent to a one-base slippage intermediate of frameshift
mutagenesis. This seems curious because there is no apparent hydrogen
bonding in the template-primer that would stabilize positioning of the 5
base at the template site. One possibility is that the PdG residue
loops out of the template position and interacts with a residue on the
enzyme. This interaction might stabilize the loop in the
template-primer structure and position the 5
base at the template
site. However, another possibility is that the PdG residue does not
loop out but the binding of the template strand to pol
positions
the 5
base close enough to the template site to hydrogen bond with the
incoming dNTP. Hydrogen bonding to the incoming dNTP would help
stabilize the slippage structure. This is essentially the mechanism
recently proposed by Efrati et al. (37) to explain the
pattern of dNMP incorporation opposite abasic sites by pol
. These
investigators found that pol
utilizes the base 5
to the abasic
site as the template for dNMP incorporation in a manner analogous to
that which we found for PdG. They term this bypass by dNTP
stabilization (37).
The structural basis for the dNTP stabilization mechanism is provided
by recent studies of the crystal structure of pol (39). Pelletier
et al. (39) proposed a model for the interaction of pol
with template-primers during gap filling synthesis which posits that
the single-stranded template is bent 90° to span the distance between
the catalytic domain and an 8-kDa domain that binds to the phosphate
group at the 5
end of the gap (39, 42). Model building suggests that
as the template strand is bent, the base 5
to the normal template base
moves closer to the template site and rotates toward the incoming base.
Although the present study did not employ gapped DNA substrates, the
8-kDa domain of pol
also has been shown to bind to the
single-stranded template overhang (43). Bending of the template strand
by pol
would move the base 5
to PdG into a position where it could
be used as template preferentially to PdG, which contains relatively
poor hydrogen bonding capacity.
Once a dNMP is inserted opposite the base 5 to PdG, continued
synthesis to the end of the template would produce one-base-deleted products. In fact, these deletion products are seen with all
template-primers extended by pol
regardless of sequence context. To
generate full-length extension products, pol
must permit slippage
of the template-primer so the newly inserted base is opposite PdG. Extension to the end of the template then produces a full-length product. Slippage may occur on the enzyme or following dissociation of
the enzyme from the template-primer. Pol
is not a highly processive
polymerase even when it is acting on undamaged template-primers so
dissociation and reassociation with PdG-containing template-primers should occur readily (33). A similar observation also has been reported
for an undamaged template in which a T
G transversion occurred at
the 5
most T in the sequence 5
-CGTTTTAC (44). This base pair
subtitution was explained by a dislocation mutagenesis model (44).
In our studies, full-length extension was seen with template-primers
containing A, G, or T residues 5 to PdG but not with a template-primer
containing a C residue 5
to PdG. Since the relative yield of deletion
and full-length extension products requires partitioning of the slipped
intermediate produced by base insertion opposite the 5
base, the rate
of slippage of the intermediate containing a base pair between a newly
inserted G and the C 5
to PdG may be sufficiently slow to preclude
slippage to an intermediate that can be extended to full-length. This
would result in extension of only the slipped intermediate to produce a
one-base-deleted product.
Pol has the ability to bind to and extend slippage intermediates
with loops containing more than one base (45). For example, a four-base
deletion product was detected in our studies when the base 5
to PdG
was G. Mechanistic studies suggested that this product arose by 1)
insertion of dCMP opposite the 5
G; 2) slippage to form a four-base
loop; and 3) extension. To our knowledge, this is the first report
where a four-base-deleted product has been characterized by DNA
sequence analysis and site-specific mutation. A similar four-base
deletion product was not detected in reactions catalyzed by the Klenow
fragment. This difference in the ability to catalyze deletions in
vitro may result from differences in the ability of the two
enzymes to accommodate extrahelical loops or bends in their binding
sites. The structure of a duplex oligonucleotide containing PdG in a
four-base loop has not been determined but Stone and co-workers (46,
47) have determined the structure of a duplex containing a PdG-C loop.
In fact, the DNA molecule does not exist as a duplex with an
extrahelical loop but as two duplex segments bent at approximately a
25-35° angle with respect to each other and connected by the PdG-C
strand (47). Similar structures may bind to the active sites of the
Klenow fragment and pol
because both enzymes catalyze two-base
deletions in vitro. However, the ability of pol
to
generate four-base deletions may reflect its ability to bend the DNA
molecules by up to 90° which would permit binding of orthogonally
oriented duplex segments connected by single strands longer than two
bases.
The results of our experiments with PdG do not appear to be general for
exocyclic adducts with blocked Watson-Crick base pairing regions.
Shibutani et al. (48) recently reported on the miscoding properties of another exocyclic adduct,
3,N4-etheno-2-deoxycytidine (
dC), in
DNA synthesis catalyzed by pol
and other mammalian polymerases.
They found that pol
incorporated dNMP's opposite
dC in the
order dCMP > dAMP > dTMP. Although the effect of sequence
context was not explored, the base 5
to
dC in the template strand
was C so one would have predicted preferential incorporation of dGMP
opposite
dC if template-slipping occurred analogous to what we
observed for PdG. The contrast between the results with
dC and PdG
may relate to the ease with which the two adducts are bypassed by pol
. Template-primers containing
dC are completely extended to
full-length products on incubation at 25 °C for 60 min with 0.5-1
units of enzyme; base-deleted products comprise less than 1% of the
extension products (48). In contrast, PdG-containing template-primers
incubated at 25 °C for 30 min with 4 units of enzyme were extended
by only 2-3% and base-deleted products comprised the majority of the
products in most sequence contexts. This suggests that PdG is a much
more powerful block to replication than
dG and that template
slippage as indicated in Fig. 11 is required for PdG bypass. The study
by Shibutani et al. (48) establishes that
dC provides
hydrogen bonding information to direct deoxynucleoside monophosphate
incorporation and that
dC:N base pairs can be formed and extended
without the necessity for template slippage. PdG appears to have no
ability to direct the incorporation of dNMP's when it is acted upon by
pol
so slippage is required for bypass. Pol
may be especially
sensitive to replication blockade by exocyclic adducts like PdG because it does not act efficiently on template-primers containing
single-stranded templates even when they contain adducts, such as
O6-methylguanine, that are readily bypassed by
other polymerases (49, 50).
The similarity of the results of our experiments with PdG to those of
Efrati et al. (37) with abasic sites suggest that PdG is
functionally a non-informational lesion when it is being acted upon by
pol . However, this classification is not general for PdG
replication by all DNA polymerases. For example, the Klenow fragment
preferentially incorporates purines opposite PdG when it is replicating
PdG-containing template-primers (20, 21). The ratio of dGMP to dAMP
inserted depends upon the identity of the base 3
to PdG in the
template strand. No dependence of the deoxynucleoside
monophosphate incorporated on the identity of the 5
base was
observed. Interestingly, although both dGMP and dAMP were
incorporated opposite PdG, only template-primers containing dAMP
opposite PdG were extended to full-length (20). Thus, PdG provides
"information" for both base incorporation and extension when it is
being acted upon by Klenow fragment but not in DNA replication by pol
.
Finally, it is important to note that the present observations provide
support for a mechanism of adduct-induced frameshift mutagenesis in
which template slippage precedes deoxynucleoside monophosphate
incorporation. Previous studies by Grollman and associates (21) have
established that frameshift mutations generated during in
vitro DNA replication by the Klenow fragment arise by a
dislocation mechanism in which bases are incorporated opposite the
adduct followed by template slippage to form a base pair between the
inserted base and a base in the template strand on the 5 side of the
lesion. This mechanism was observed with several different adducts
including PdG (21). Previous work from our laboratory has confirmed
this finding for DNA replication by Klenow fragment of template-primers
containing PdG in different sequence contexts (20). However, our
present findings indicate that pol
introduces frameshifts by a
different mechanism, one in which template slippage precedes
deoxynucleoside monophosphate incorporation. Thus, two different
enzymes, E. coli DNA polymerase I and human DNA polymerase
acting on the same adduct in the same template-primer introduce frameshift mutations by opposite mechanisms.
We are grateful to Sam Wilson for a helpful discussion.