From the Departments of Biological Sciences and
Chemistry, Hedco Molecular Biology Laboratories, University of Southern
California, Los Angeles, California 90089-1340, the
§ European Molecular Biology Organization, Heidelberg 69012, Germany, and the ¶ National Institute of Environmental Health
Sciences, Research Triangle Park, North Carolina 27709
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), a
common oxidative DNA lesion, favors a syn-conformation in
DNA, enabling formation of stable 8-oxo-dG·A base mispairs resulting
in G·C A significant increase in G·C Procaryotic and eucaryotic cells contain highly conserved groups of
enzymes to deal with the potentially lethal levels of 8-oxo-dG-stimulated mutations. Analogs of Escherichia coli
MutM glycosylase excise 8-oxo-dG from DNA (paired opposite C) (6), whereas the MutY class of enzymes remove dAMP paired opposite 8-oxo-dG
(1). Taken together, MutM and MutY, along with MutT, responsible for
eliminating oxidized G from deoxy- and ribonucleoside triphosphate
pools (7, 8), constitute the "8-oxo-dG repair system" (reviewed in
Ref. 9). Although most 8-oxo-dG lesions are repaired by this system,
those that escape repair are likely to encounter DNA polymerases during
either replicative or repair DNA synthesis.
The nucleotide incorporation specificity opposite 8-oxo-dG may vary
depending on which polymerase is used to copy the lesion in
vitro (10). Furthermore, very little is known regarding the effects of sequence context on incorporation specificities for different polymerases (11). In this paper, we have used human DNA
polymerase (pol) Materials
Enzymes--
Human pol Nucleotides--
Nonradioactive nucleotides were purchased from
Pharmacia Biotech Inc. [ Synthesis of DNA Primer-Templates--
8-Oxo-dG phosphoramidite
was synthesized as described (14). Templates containing a single
8-oxo-dG site, control templates with no lesion, and various primers
were synthesized on an Applied Biosystems 392 DNA/RNA synthesizer.
Oligodeoxyribonucleotides containing a 5'-phosphate group, needed to
form 5-base gaps for pol DNA Substrates--
To investigate the insertion specificity of
pol Methods
Conditions for Determining Kinetic Constants--
Primers were
5'-end-labeled with 33P using T4 polynucleotide kinase as
described (16). Annealing was carried out by incubating 7.5 nM primer with 7.5 nM template and 38 nM downstream oligodeoxyribonucleotide in pol Kinetics of Insertion Opposite 8-Oxo-dG--
Time courses were
run to establish the conditions for measuring incorporation
efficiencies opposite 8-oxo-dG. Reactions were carried out by mixing
equal volumes of annealed primer-template with reaction buffer
containing deoxyribonucleotide (for buffer components, see annealing
conditions described above). The final concentration of primer-template
in the reactions was 3.8 nM. pol
Four primer-template constructs were used to investigate the
effect of the downstream nearest neighbor on incorporation efficiencies at the 8-oxo-dG site. For each reaction, the dNTP substrate for incorporation opposite the lesion was varied in concentration over a
range of 10 µM to 1.2 mM. In the time course
experiments, the dNTP substrate was held constant at 800 µM. The reactions were performed under running-start
conditions with 5 µM dTTP for insertion opposite the two
As upstream from 8-oxo-dG. Reactions were terminated by adding 2 volumes of 20 mM EDTA in 95% formamide and heating to
100 °C for 5 min. Samples were then cooled on ice, and the
single-stranded primers were separated according to length by
electrophoresis on 18% polyacrylamide gels.
The kinetics of incorporation of each of the four deoxynucleotides
opposite the lesion were determined as a function of deoxynucleotide concentration (17, 18). Band intensities were measured by a
PhosphorImager using Imagequant software (Molecular Dynamics, Inc.,
Sunnyvale, CA). Incorporation efficiencies were established as
described (16).
Dideoxynucleotide Incorporation--
To determine the nature of
the dNMPs incorporated at or around the lesion site by pol Sequence Markers--
We identified each product sequence based
on the dideoxynucleotide sequencing data. An independent sequence
confirmation was obtained first by synthesizing each "full size"
oligonucleotide sequence on a DNA synthesizer (Applied Biosystems 392 DNA/RNA synthesizer). The oligonucleotides were gel-purified and
5'-end-labeled with 33P. A sequencing ladder containing a
digest of the labeled oligonucleotides was obtained by
3'-exonucleolytic digestion using E. coli DNA polymerase III
core (containing the We have measured the effects of sequence context on dAMP and dCMP
insertion efficiencies opposite 8-oxo-dG using human polymerases T·A transversion mutations. When human DNA polymerase
(pol)
was used to copy a short single-stranded gap containing a
site-directed 8-oxo-dG lesion, incorporation of dAMP opposite 8-oxo-dG
was slightly favored over dCMP depending on "downstream" sequence
context. Unexpectedly, however, a significant increase in dCMP·A and
dGMP·A mispairs was also observed at the "upstream"
3'-template site adjacent to the lesion. Errors at these undamaged
template sites occurred in four sequence contexts with both gapped and
primed single-stranded DNA templates, but not when pol
replaced pol
. Error rates at sites adjacent to 8-oxo-dG were roughly 1% of the
values opposite 8-oxo-dG, potentially generating tandem mutations during in vivo short-gap repair synthesis by pol
. When
8-oxo-dG was replaced with 8-bromo-2'-deoxyguanosine, incorporation of dCMP was strongly favored by both enzymes, with no detectable misincorporation occurring at neighboring template sites.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
T·A mutation rates (1) takes
place when oxidative damage to DNA occurs at C-8 of G to form 8-oxo-dG1 (2, 3). In contrast
to G, which exists predominantly in an anti-conformation in
DNA and forms base pairs almost exclusively with C, base pairing
properties of 8-oxo-dG tend to be ambiguous, having a
syn-conformation when paired opposite A (4) and an anti-conformation when paired opposite C (5).
and pol
to investigate specificity of incorporation opposite 8-oxo-dG, varying the template base immediately downstream from the lesion. During the course of this study, we observed unexpectedly that in addition to its own ambiguous base pairing properties, 8-oxo-dG also stimulates nucleotide
misincorporations at adjacent upsteam and downstream
template sites. This paper is focused on the potential implications of
8-oxo-dG-stimulated "action-at-a-distance" mutagenesis in relation
to pol
-catalyzed gap repair synthesis.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was purified as described (12).
pol
(2000 units/mg), a kind gift from Dr. T. S.-F. Wang,
refers to the large catalytic subunit of the holoenzyme and was
purified as described (13). Both enzymes are highly purified and
contain no detectable 3'-exonuclease activity. One DNA polymerase unit
is defined as the incorporation of 1 nmol of dNMP into DNA in 1 h
at 37 °C. DNA polymerase activity for pol
was measured on gapped
primer-template DNA and for pol
on ungapped DNA. T4 polynucleotide
kinase was purchased from U. S. Biochemical Corp.
-33P]ATP (2000 Ci/mmol) was
purchased from NEN Radiochemicals.
, were synthesized by Lynn Williams at the
University of Southern California Comprehensive Cancer Center.
opposite 8-oxo-dG, 5-base gaps with the lesion in their center
were formed by annealing 15-mer primers and 22-mer downstream
oligodeoxyribonucleotides to 42-mer templates with a 8-oxo-dG lesion at
position 18. The downstream oligodeoxyribonucleotides were
phosphorylated at their 5'-end. This and a gap size of fewer than 6 nucleotides are required by pol
for processivity (15). For studies
of insertion opposite normal G or 8-Br-dG, similar primer templates
were used with either G or 8-Br-dG substituting for 8-oxo-dG at
position 18. The same experiments were also carried out with pol
,
but no downstream oligodeoxyribonucleotides were used since these
inhibited pol
activity ~2-fold (data not shown). All primers and
templates were purified by polyacrylamide gel electrophoresis.
reaction
buffer (35 mM Tris-HCl, pH 8.0, 6.7 mM
MgCl2, 100 mM NaCl, 0.2 mg/ml bovine serum
albumin, 1.5 mM dithiothreitol, and 2% glycerol) at
37 °C for 1 h, followed by gradual cooling to room temperature.
For pol
, 7.5 nM primer was annealed to 7.5 nM template in pol
reaction buffer (20 mM
Tris-HCl, pH 8.0, 10 mM MgCl2, 0.2 mg/ml bovine
serum albumin, and 1 mM
-mercaptoethanol) at 37 °C
for 1 h, followed by gradual cooling to room temperature.
(0.2 units) or pol
(0.02 units) was added, and the reactions were incubated at 37 °C
for 10 min. Under these conditions, the data for both enzymes were in
the linear range.
, primer
extension reactions were carried out in the presence of 5 µM dTTP (running-start base) and a 50 µM
concentration of either dATP or dCTP. At various time points (a range
of 1-30 min), ddATP or ddCTP was added to a final concentration of 0.5 mM, and the reactions were allowed to continue for an
additional 5 min at 37 °C. Reactions were terminated as described
above. Controls were run under the same conditions, but in the absence of dideoxynucleotides. Reaction products were separated on 18% polyacrylamide gels.
-proofreading exonuclease) under the following
conditions. Labeled oligonucleotide (13 nM) was incubated
with 0.5 nM pol III core for 2-5 min at 37 °C in pol
III reaction buffer (20 mM Tris-Cl, pH 7.5, 40 µg/ml
bovine serum albumin, 50 mM dithiothreitol, 50 mM NaCl, and 8 mM MgCl2). Reactions
were quenched as described above. Samples were run alongside pol
reaction products on 18% polyacrylamide gels.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
. Synthesis by pol
involves filling in a 5-nucleotide template gap, 3'-AA 8-oxo-dG NC-5', where the nucleotide N,
immediately downstream from the lesion, is varied (Fig.
1 and Table
I). Two Ts are incorporated opposite the
two running-start As, followed by incorporation of each of the four
dNMPs opposite 8-oxo-dG. We have also used pol
to copy a subset
of these sequences (Table I).
View larger version (61K):
[in a new window]
Fig. 1.
Occurrence of doublet bands when pol
is used to copy template containing 8-oxo-dG
lesions in different sequence contexts. Incorporation of dNMPs
opposite 8-oxo-dG (GO) was measured at the following dNTP
concentrations: a, 10, 25, 100, 200, 600, and 1200 µM dATP or dCTP and 600 and 1200 µM dGTP or
dTTP; b, 10, 25, 100, 200, 600, and 1200 µM
dATP or dCTP and 10, 200, and 600 µM dGTP or dTTP;
c, 25, 100, 200, 600 and 1200 µM dATP and 10, 25, 100, 200, 600, and 1200 µM dCTP; d, 10, 25, 100, 200, 600, and 1200 µM dATP and 10, 100, 200, 600, and 1200 µM dCTP. dTTP (5 µM) was
included in running-start reactions to extend the primer 3'-end to
reach 8-oxo-dG. Template sequences are shown at the left.
Arrows shown at the right indicate the locations of doublet
bands. Reactions were carried out as described under "Experimental
Procedures."
Nucleotide incorporation specificities for pol and
opposite
8-oxo-dG, 8-Br-dG, and G
Nucleotide Incorporation Specificities Opposite 8-Oxo-dG--
An
analysis of the data reveals that pol inserts dAMP slightly more
efficiently than dCMP opposite 8-oxo-dG with three of the four
5'-nearest-neighbor bases and inserts dAMP and dCMP with about equal
efficiency with the other nearest-neighbor base (G) (Table I). Both
dAMP and dCMP are incorporated slightly more efficiently when C is the
5'-nearest neighbor. The efficiency of dCMP incorporation opposite
8-oxo-dG is 4-fold less than that of dCMP incorporation opposite G in
this sequence context. This same template sequence, having an abasic
site in place of 8-oxo-dG, also showed the highest misincorporation
efficiency (16). Low intensity gel bands corresponding to the
incorporation of either dGMP or dTMP opposite 8-oxo-dG are clearly
visible (Fig. 1, a and b). However, their
corresponding Vmax/Km values
are <1% of the values for incorporation of either A or C and are
excluded from Table I. pol
also preferentially incorporates A
opposite 8-oxo-dG. However, in contrast to pol
, which incorporates
A with at most a 2-fold higher efficiency compared with C, pol
favors incorporation of A with much higher selectivity, ~5-10-fold (Table I).
8-Oxo-dG-stimulated Nucleotide Misincorporation at Neighboring
Template Sites Catalyzed by pol --
The gel band patterns in Fig.
1 demonstrate that dAMP and dCMP are both readily incorporated opposite
8-oxo-dG by pol
and are also extended 1 nucleotide beyond the
lesion by incorporation of a mismatched base pair. Remarkably, however,
in addition to its ambiguous base pairing and promiscuous extension
properties, 8-oxo-dG also stimulates nucleotide misincorporations at
adjacent upstream template sites. Correct and incorrect incorporations opposite a single template site appear as doublet gel bands (Fig. 1).
When a combination of dTTP (at 5 µM, for incorporation
opposite the running-start As) and either dCTP or dGTP at increasing
concentrations is included in pol
reactions, doublet bands occur
upstream, downstream, or directly opposite 8-oxo-dG in a
sequence-dependent manner. In contrast, there are no
doublet bands detected when dATP is used as a substrate for pol
,
even when high-resolution (20% polyacrylamide) gels are used (data not
shown). Data illustrating the presence of doublets are presented in
which C (Fig. 1a), A (Fig. 1b), G (Fig.
1c), or T (Fig. 1d) is immediately downstream from 8-oxo-dG on the template strand.
We can identify individual doublet band sequences unambiguously, in
almost all instances, using the following criteria. Dideoxynucleotides (ddCTP or ddATP) are added to the reaction at different time points, resulting in termination of primer extension following incorporation of
each ddNMP by pol . Each specific oligonucleotide sequence corresponding to the partial sequences deduced from the termination data, along with permutations of other possible sequences, are then
synthesized; the synthetic oligonucleotides are end-labeled with
33P and used as molecular mass markers for comparison with
polymerase reaction products (Fig. 2).
For example, the doublet appearing opposite template A in the
dGTP lane (Fig. 2a) contains G and T as upper and
lower bands, respectively, based on the marker digest shown to the
right. In a similar way, the doublet in the dCTP lane (Fig.
2b) is resolved into a lower C and an upper T.
|
With C as the adjacent downstream neighbor (Fig. 1a), doublets occur only when dTTP + dCTP are present in the reaction. The lesion is flanked by both upstream and downstream doublets. The upstream doublet opposite A is easily observed, even at the lowest concentration of dCTP (10 µM), and reflects the correct incorporation of dTMP (upper band in doublet) and the misincorporation of dCMP opposite A (lower band in doublet). The downstream doublet is much less intense, appearing at dCTP concentrations above 200 µM. The downstream doublet results from incorporation of two dCMPs (opposite 8-oxo-dG and then opposite C) from each upstream doublet. No other dNTP gives rise to doublets in this sequence context.
Note that the 5-nucleotide gap is filled to the end when dGTP is
present in the reaction. However, in contrast to incorporation of
either dCMP or dAMP, incorporation of dGMP is unlikely to be occurring
directly opposite 8-oxo-dG. Instead, G incorporation is likely to be
taking place opposite the template C downstream from 8-oxo-dG
(3'-AA 8-oxo-dG CC) (Fig. 1a) by "skipping
over" 8-oxo-dG using a transiently misaligned primer 3'-end (Fig.
3; see "Discussion") (16).
|
In the template sequence 3'-AA 8-oxo-dG GC (Fig. 1c), both dAMP and dCMP are incorporated almost equally, primarily because of a reduction in Vmax/Km for incorporation of dAMP directly opposite 8-oxo-dG as opposed to an increase in the dCMP incorporation efficiency, e.g. compare Vmax/Km values in the dATP and dCTP columns for the four gapped primer-template DNAs containing 8-oxo-dG (Table I, first four sequences). Note that Vmax/Km for incorporation of dCMP is no higher than for the other three sequences (Table I), suggesting that misalignment misincorporation may not be occurring.
Three doublets are observed when G is located downstream from 8-oxo-dG when the reaction is carried out in the presence of dCTP + dTTP. The doublets occur opposite both upstream running-start As and directly opposite 8-oxo-dG (Fig. 1c, dCTP lanes). Weak intensity bands running just below the bands corresponding to incorporation of each running-start T (Fig. 1c, lower two arrows) are generated by misincorporation of dCMP opposite both As upstream from 8-oxo-dG. Further extension of each of the lower doublets gives rise to weak doublet bands opposite 8-oxo-dG (Fig. 1c, upper arrow). The weak bands corresponding to incorporation of C opposite 8-oxo-dG are efficiently extended by incorporation of another dCMP opposite the downstream G, followed, most likely, by misincorporation of dCMP opposite C.
We also find that dAMP·8-oxo-dG base pairs are easily extended by two
consecutive misincorporations of dAMP opposite the template bases G and
C downstream from the lesion (Fig. 1c, dATP
lanes). The relative ease with which nucleotides can be
misincorporated by pol downstream from 8-oxo-dG (Fig.
1c, dATP and dCTP lanes) may result
from significant template distortions by 8-oxo-dG and by primer termini
distortions caused by the non-Watson-Crick 8-oxo-dG·A base pairing
structure. For the template sequence having T proximal to 8-oxo-dG,
there is a single upstream doublet when dCTP is included in the
reaction, originating from correct incorporation of dTMP and
misincorporation of dCMP opposite the second running-start A (Fig.
1d).
Since the doublets are sequence-dependent, we decided to
mask the template sequence downstream from 8-oxo-dG, by reducing the
gap length from 5 to 3 bases, to see if the upstream doublet bands were
altered by eliminating the downstream region of ssDNA. Indeed, the
doublet bands were not altered. An example is shown where the template
region to the 5'-side of 8-oxo-dG is annealed to a downstream
oligonucleotide (Fig. 4). The doublet
upstream from 8-oxo-dG is present at the same dCTP concentration (10 µM) either with or without the covered downstream Cs
(compare Figs. 4 and 1a).
|
Misincorporations Proximal to 8-Oxo-dG Are Catalyzed by pol ,
but Not by pol
--
The 8-kDa amino-terminal domain of pol
has
been shown to interact with the 5'-phosphoryl group on the downstream
gap-forming DNA when functioning as a deoxyribose phosphate lyase
during base excision repair (19, 20). Therefore, it is necessary to
determine if the doublets occur only in the presence of gapped DNA.
Since pol
is much more active and processive with primer-template DNA containing a 1-6-nucleotide gap compared with primed ssDNA (15),
we carried out reactions with both gapped and primed ssDNAs over a
10-fold range of enzyme concentration (Fig.
5a). The substrates for the
reaction, dTTP for the running start and dCTP for incorporation opposite 8-oxo-dG, are those that give rise to doublet bands opposite the upstream A proximal to 8-oxo-dG (Fig. 1a, dCTP
lanes).
|
Doublet bands opposite A are observed at each pol level in the
gap-filling reaction (Fig. 5a, right panel).
However, despite significantly reduced synthesis using primed ssDNA, a
doublet band is nevertheless detectable opposite the upstream A at the highest pol
concentration used (Fig. 5a, left
panel). Therefore, we conclude that the interaction between the
8-kDa domain of pol
and the 5'-phosphoryl is not linked exclusively
to the presence of the dCMP·A upstream misincorporation band.
Although the downstream oligonucleotide is not necessary for doublet
formation, its presence is required for continued synthesis beyond
8-oxo-dG. A similar experiment performed using pol
shows that
doublets are not observed with either primer-template DNA (Fig.
5b), suggesting that these unusual misincorporation events
are dependent not only on the presence of 8-oxo-dG, but also on
properties specific to pol
. Again, the misincorporations upstream
of 8-oxo-dG could be linked to 8-oxo-dG-induced distortions in
the template (see "Discussion").
pol -catalyzed Doublet Misincorporation Bands Require the
Presence of 8-Oxo-dG--
Templates with normal G in place of 8-oxo-dG
were incubated with pol
(Fig. 6) or
pol
(data not shown). Both polymerases incorporate two dTMPs
opposite the two running-start As and dCMP opposite G. At high dCTP
concentrations (
100 µM), pol
catalyzes weak
misincorporation of dCMP opposite the downstream C. However, no
doublets are formed by pol
using templates in which either G (Fig.
6) or T (data not shown) is used in place of 8-oxo-dG.
|
NMR studies have shown that 8-oxo-dG favors a
syn-conformation when paired opposite A (4, 21). Data
showing favored hydrolysis of both 8-oxo-dGTP (22) and 8-Br-dGTP (23)
over dGTP by the MutT hydrolase suggest that 8-Br-dG also favors a
syn-conformation (24), although direct NMR evidence is not
yet available to substantiate this point. To investigate whether a
syn-nucleotide is required to generate mispair-containing
doublet bands proximal to the template lesion, 8-Br-dG was substituted
for 8-oxo-dG and copied using reactions similar to those displayed in
Fig. 1a (with C as the 5'-neighbor). Reactions were carried
out using pol (Fig. 7a) and pol
(Fig. 7b).
|
Both polymerases exhibit similar primer extension patterns in the presence of 8-Br-dG. Following incorporation of the running-start Ts, dCMP and dAMP are incorporated directly opposite the modified template base. However, in contrast to the templates containing 8-oxo-dG, for which incorporation of dAMP is favored over dCMP (Table I), the reverse is now true, with dCMP strongly favored for incorporation opposite 8-Br-dG by both polymerases (Fig. 7, a and b). Note that, as before, the highly efficient incorporation of dGMP is unlikely to be taking place directly opposite the lesion, but more likely results from pairing an incoming dGTP opposite a downstream template C using a dNTP-stabilized misalignment mechanism (Fig. 3).
The most important point is that the doublet patterns so obvious with
8-oxo-dG in this sequence context (Fig. 1a) do not occur with 8-Br-dG. Thus, the emergence of upstream doublet bands when synthesis is carried out using pol appears to require the presence of 8-oxo-dG on the template strand.
Doublets Can Be Driven Out by Adding the dNTP Complementary to the
Downstream Neighbor of 8-Oxo-dG--
In an attempt to drive out the
upstream doublet on the template with C located 5' to 8-oxo-dG (Fig.
1a), incubations were carried out using three dNTPs: dTTP
for the running start, a high concentration of dCTP (1.2 mM) to form the doublet, along with increasing
concentrations of dGTP, complementary to the downstream Cs (Fig. 8).
The doublet is clearly visible in the absence of dGTP (Fig.
8, first lane). However, as
the concentration of dGTP is increased, the lower band of the doublet,
corresponding to misincorporation of dCMP opposite the second
running-start A, diminishes in intensity concomitant with complete
filling of the 5-nucleotide gap (Fig. 8, dGTP lanes). We
suggest that the dCMP·A mispair is eliminated because the binding of
dCTP on the polymerase-primer-template DNA complex is reduced by
mass-action competition with dGTP. However, in contrast to
8-oxo-dG-stimulated dCMP·A mispair formation, 8-oxo-dG does not
stimulate misincorporation of dGMP opposite the upstream A.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
8-oxo-dG DNA template lesions are believed to contribute
significantly to the large increase in mutation rates observed in oxidatively damaged cells. NMR studies have shown that 8-oxo-dG can
assume two conformations, a favored syn-conformation, in
which base mispairs are formed with A (4), or an
anti-conformation, forming "correct" base pairs with C
(21). Previous measurements using DNA polymerase in vitro
(10), including this investigation of the effect of sequence context on
the specificity of incorporation by pol opposite 8-oxo-dG (Fig. 1),
confirm that both dAMP and dCMP are incorporated, suggesting that
8-oxo-dG exhibits both anti- and syn-conformation
base pairing properties during replication.
8-Oxo-dG Reduces pol Fidelity at Neighboring Template
Sites--
When synthesis is carried out using pol
, the data
reveal an interesting and unexpected gel band pattern, namely the
appearance of doublet bands at template sites adjacent to 8-oxo-dG
(Fig. 1). A doublet band upstream from 8-oxo-dG corresponds to
incorporation of a different nucleotide prior to reaching
the lesion site and thus reflects the ability of 8-oxo-dG to stimulate
misincorporation errors at neighboring template sites.
The doublet bands upstream from 8-oxo-dG vary, depending on the sequence downstream from the lesion. In the sequence 3'-AA 8-oxo-dG CC, an upstream doublet opposite A occurs, generated by a correct incorporation of dTMP opposite A and a misincorporation of dCMP opposite A (Fig. 1a). Extension of each of the doublets by incorporation of dCMP opposite 8-oxo-dG followed by misincorporation of dCMP opposite C also gives rise to downstream doublet bands.
The nature of the doublets change for the sequence 3'-AA 8-oxo-dG AC,
where a doublet is formed by correct incorporation of dTMP and
misincorporation of dGMP opposite the upstream running-start A (Fig.
1b). For the sequence 3'-AA 8-oxo-dG GC, doublets
corresponding to dCMP misincorporation (and correct dTMP incorporation)
are observed opposite both upstream running-start As, and a
doublet band is also formed opposite 8-oxo-dG (Fig. 1c). For
the sequence 3'-AA 8-oxo-dG TC, a doublet occurs opposite the
upstream A, corresponding, as before, to incorporation of dTMP and dCMP
(Fig. 1d). This action-at-a-distance effect appears to be a
novel property of the interaction of pol with 8-oxo-dG, reducing
the fidelity of DNA synthesis at neighboring template positions while,
at the same time, resulting in mutations at the site of the lesion.
Doublet Band Dependence on 8-Oxo-dG and pol --
Doublets are
not observed in the following three series of experiments.
(i) Synthesis carried out by pol
on either gapped or primed ssDNA
fails to give rise to doublet bands adjacent to 8-oxo-dG, even when
high levels of enzyme are used (Fig. 5b). Indeed, none of
the 8-oxo-dG sequence contexts giving rise to doublets when copied by
pol
results in detectable doublet bands using pol
(data not
shown), suggesting that these bands arise from a specific interaction
between pol
and 8-oxo-dG, as opposed to the presence of base
composition heterogeneity in the synthetic templates. (ii) There are no
doublet bands observed when 8-oxo-dG is replaced by G (Fig. 6) or T
(data not shown) and copied with pol
. Thus, the presence of pol
alone is insufficient to generate doublets. (iii) No doublets are
observed when 8-oxo-dG is replaced by 8-Br-dG and copied with pol
(Fig. 7a). 8-Br-dG, like 8-oxo-dG, is believed to favor a
syn-conformation. Thus, it appears that substitutions
at the C-8 atom on the purine ring causing a 180° rotation about the
glycosidic bond might not be the sole basis for misincorporations
occurring at neighboring template positions, but that some additional
property of 8-oxo-dG is also required. For example, should the C-8
substituted purine ring adopt an anti-conformation, the
significant steric hindrance with the template phosphodiester backbone
could be a cause of the misincorporations at neighboring template bases.
The 8-kDa domain of pol is in direct contact with the 5'-phosphoryl
group on the downstream portion of the gap, giving rise to a relatively
processive gap-filling reaction (15). pol
is much more active on
DNA containing short, 1-6 nucleotide gaps (15, 25) than on primed
ssDNA templates, as confirmed in Fig. 5a. However, an
upstream doublet band is still observed when synthesis is carried out
at a high concentration of pol
on primed ssDNA (Fig. 5a,
left panel), despite its reduced activity on ungapped DNA.
Therefore, the doublet bands are not solely attributable to protein-DNA
interactions involving the 8-kDa domain taking place downstream from
8-oxo-dG. Doublets are not observed using levels of pol
comparable
to pol
on either gapped or ungapped DNA (Fig. 5b).
Sequence Context Dependence of Nucleotide Misincorporation Directly
Opposite 8-Oxo-dG--
The biological significance of 8-oxo-dG lies in
its ability to cause a sizable increase in mutation rates caused by
preferential base pairing with A rather than C (9). In contrast to pol
, which strongly favors the incorporation of A opposite 8-oxo-dG (Table I) (10), pol
is much more "even-handed" in the four sequence contexts examined, favoring the incorporation of A compared with C opposite 8-oxo-dG by no more than 2.4-fold (Table I).
In the sequence 3'-AA 8-oxo-dG GC, we expected that dCMP
incorporation would be strongly enhanced by skipping past 8-oxo-dG and
pairing directly with the nearest-neighbor downstream G, owing to the
propensity of pol to copy past lesions via dNTP-stabilized misalignment (Fig. 3) (16, 26). However, this was not the case since
there is an ~2-fold reduction in the incorporation rate of
dCMP (and also of dAMP) compared with the 3'-AA 8-oxo-dG CC sequence.
Although transient primer-template DNA misalignment may be occurring,
we cannot distinguish this mode unambiguously from direct incorporation
of dCMP opposite 8-oxo-dG followed by extension opposite the downstream
G site.
However, evidence that pol can carry out dNTP-stabilized
misalignment synthesis is found with the sequence 3'-AA 8-oxo-dG CC
(Fig. 1a, dGTP lanes). Here, incorporation of
dGMP is unlikely to occur directly opposite 8-oxo-dG, but is instead
taking place 1 base downstream from 8-oxo-dG, opposite C. In support of
this model, we find that extension beyond the lesion using dGTP as substrate is reduced significantly when A is the template base downstream from 8-oxo-dG (3'-AA 8-oxo-dG AC) (Fig.
1b, dGTP lanes), whereas extension past 8-oxo-dG
does take place using dTTP as substrate. At high dGTP concentration
(600 µM), however, trace bands can be detected
corresponding to misincorporation of dGMP directly opposite 8-oxo-dG
followed by correct additions of T and G opposite the next 2 downstream
template bases. A single upstream doublet, occurring only at high dGTP
concentrations (200 and 600 µM) when A is located 5' to
8-oxo-dG (Fig. 1b), is attributable to formation of a
dGMP·A mispair.
Comparison with Previous in Vitro Data Using Polymerases to Copy
8-oxo-dG and Possible Biological Consequences--
In an earlier
in vitro study, Shibutani et al. (10) reported
that pol strongly favors formation of dAMP·8-oxo-dG over dCMP·8-oxo-dG mispairs by a ratio of 200:1. Our data using pol
also demonstrate preferential incorporation of dAMP opposite 8-oxo-dG,
but with a lower specificity of ~10:1. For pol
, we find that
incorporation of A is only slightly favored over C in the four sequence
contexts examined.
In contrast to our data, Shibutani et al. (10) found a
reversal in the specificity of pol favoring dCMP·8-oxo-dG over dAMP·8-oxo-dG by 4:1, although kinetic data for pol
were not reported. As mentioned above, the activity of pol
is much greater when filling a short gap than when extending primed ssDNA (see, for
example, Fig. 5 and Table I). The previous measurements were made using
primed ssDNA (10), whereas we focused our attention on short
gap-filling reactions (Fig. 1), which are believed to be most relevant
for base excision and short nucleotide excision repair in
vivo (27, 28). However, similar measurements using pol
to copy
primed ssDNA gave a dAMP/dCMP incorporation efficiency ratio of ~1.8
(Table I), which is similar to the incorporation efficiencies found for
gapped DNA. As expected, the activity of pol
was much greater
(~200-fold) using gapped primer-template DNA. Because the 18-mer
template used by Shibutani et al. (10) contained no Gs
downstream of 8-oxo-dG, the preference for C cannot be attributed to
enhanced dCMP incorporation by transient primer-template DNA
misalignment. A difference in the primer-template sequence context
remains the most likely explanation for the differences between our
data and those of Shibutani et al. (10).
Sequence context-dependent errors occurring at residues
adjacent to 8-oxo-dG were reported by Kuchino et al.
(29) using pol I Klenow fragment. They also reported that
8-oxo-dG-directed misincorporations occurred at similar frequencies for
the four dNTPs. Shibutani et al. (10) challenged both
observations, noting that Klenow fragment was not suitable for the
dideoxynucleoside sequencing analysis used by Kuchino et al.
(29). Although our data do not speak directly to these issues, we find
that dCMP is incorporated opposite 8-oxo-dG with either pol or
.
The pol -mediated incorporation of C opposite 8-oxo-dG observed here
and by others (10) suggests that this enzyme can readily tolerate a
template DNA structure with the anti-conformer of 8-oxo-dG, which supports normal Watson-Crick base pairing with C. In this case,
the phosphate group in the phosphodiester backbone adjacent to C-8 of
8-oxo-dG must be displaced significantly (~2.7 Å), thus distorting
the template strand (data not shown). The extent of this distortion and
the ability of the neighboring base sequences to tolerate it appear to
a shared property of pol
and the sequence context, which is absent
with pol
. The presence of highly localized DNA distortions in the
active cleft of pol
may provide an underlying basis for the
action-at-a-distance mutagenesis observed here with templates
containing 8-oxo-dG.
In an experiment by Feig and Loeb (30), pol was used to copy oxygen
free radical-damaged DNA containing the gene for lacZ
. The damaged DNA was then transfected into E. coli to obtain
a lacZ
mutational spectrum, which contained three tandem
A
G transitional hot spots located 1 base upstream from a
putatively damaged G. It is likely that this class of pol
-catalyzed
mutations occurs as a result of the 8-oxo-dG-stimulated dCMP·A
mispairs that we have observed (Fig. 1).
To address the potential biological significance of misincorporations
at sites adjacent to 8-oxo-dG, we estimated
Vmax/Km values for dCMP·A
misincorporation at the template site upstream from 8-oxo-dG (Fig.
1a, lower band). We found that the
misincorporation rate at the neighboring upstream A site was ~1% of
the rate of incorporating dAMP directly opposite 8-oxo-dG, which is, in
turn, roughly 46% of the rate of correctly incorporating dCMP opposite G. It is known that 8-oxo-dG-induced mutation rates are increased significantly in the absence of the 8-oxo-dG repair pathway (9). We can
therefore estimate that mutations at sites adjacent to 8-oxo-dG could
be elevated over spontaneous levels by between 2- and 20-fold. This
action-at-a-distance effect appears to be a novel property of 8-oxo-dG
when copied specifically by pol . Thus, in addition to stimulating
G·C
T·A mutations at the site of the lesion, 8-oxo-dG might
also cause an increase in mismatch formation at neighboring template
positions within short repair tracts filled in by pol
. If such
complex mismatches were to occur in vivo, they would be
likely to serve as candidates for removal by the mammalian long-patch
post-replication mismatch repair system (31) and might therefore be
discernible in cells deficient in mismatch repair.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Joe Krahn for discussions on the
structures of pol and 8-oxo-dG.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM21422.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: Dept. of
Biological Sciences; University of Southern California, University
Park, Los Angeles, CA 90089-1340. Tel.: 213-740-5190; Fax:
213-740-8631; E-mail: mgoodman{at}mizar.usc.edu.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: 8-oxo-dG, 8-oxo-7,8-dihydro-2'-deoxyguanosine; 8-Br-dG, 8-bromo-2'-deoxyguanosine; pol, DNA polymerase; ssDNA, single-stranded DNA.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|