(Received for publication, August 12, 1996, and in revised form, November 6, 1996)
From the Department of Biological Sciences, Hedco
Molecular Biology Laboratories, University of Southern California, Los
Angeles, California 90089-1340, the § European Molecular
Biology Organization, Heidelberg, Germany 69012, and the
¶ University of Texas Medical Branch at Galveston,
Galveston, Texas 77555-1068
The "A-rule" reflects the preferred
incorporation of dAMP opposite abasic lesions in Escherichia coli
in vivo. DNA polymerases (pol) from procaryotic and eucaryotic
organisms incorporate nucleotides opposite abasic lesions in accordance
with the A-rule. However, recent in vivo data demonstrate
that A is not preferentially incorporated opposite abasic lesions in
eucaryotes. Purified human DNA polymerases and
are used to
measure the specificity of nucleotide incorporation at a site-directed
tetrahydrofuran abasic lesion, in 8-sequence contexts, varying upstream
and downstream bases adjacent to the lesion. Extension past the lesion
is measured in 4 sequence contexts, varying the downstream template
base. Pol
strongly favors incorporation of dAMP directly opposite
the lesion. In marked contrast, pol
violates the A-rule for
incorporation directly opposite the lesion. In addition to
incorporation taking place directly opposite the lesion, we also
analyze misalignment incorporation directed by a template base
downstream from the lesion. Lesion bypass by pol
occurs
predominantly by "skipping over" the lesion, by insertion of a
nucleotide complementary to an adjacent downstream template site.
Misalignment incorporation for pol
occurs by a novel
"dNTP-stabilized" mechanism resulting in both deletion and base
substitution errors. In contrast, pol
shows no propensity for this
type of synthesis. The misaligned DNA structures generated during
dNTP-stabilized lesion bypass do not conform to misaligned structures
reported previously.
Loss of purine and pyrimidine bases is a significant source of DNA damage in procaryotic and eucaryotic organisms. Abasic (apurinic and apyrimidinic) lesions occur spontaneously in DNA; in eucaryotes it has been estimated that about 104 depurination and 102 depyrimidation events occur per day (1). An equally important source of abasic DNA lesions results from the action of DNA glycosylases, most notably uracil glycosylase which excises uracil arising primarily from spontaneous deamination of cytosine (1, 2). A DNA template with an abasic lesion presents a strong block to DNA synthesis in Escherichia coli, which can be partially alleviated by induction of the SOS response (3). Although most abasic lesions are removed by a base excision repair pathway involving the combined action of various enzymes, e.g. N-glycosylase, endonuclease, phosphodeoxyribosyl phosphodiesterase, polymerase and ligase (4), a small fraction of lesions persist and are replicated leading to a high probability of a mutation occurring. When an abasic lesion is copied in vivo in E. coli, A is predominantly incorporated opposite the lesion (3, 5).
Preferential incorporation of A opposite abasic lesions is referred to as the "A-rule" (5-8). In agreement with E. coli data (3), studies with purified DNA polymerases showed that incorporation of A opposite X1 occurs about 10-fold more frequently than G opposite the lesion and about 50-fold more frequently than either C or T (9, 10). Although neighboring sequence contexts have been observed to perturb relative nucleotide incorporation efficiencies (3, 9), the strong conclusion, until recently, remained that DNA polymerases and reverse transcriptases generally behave in accordance with the A-rule. A physically intuitive basis for the A-rule emanates from NMR studies showing that when A is opposite an abasic lesion it stacks in an intrahelical configuration causing virtually no distortion of the DNA helix (11, 12). For the other bases, G may either be inter- or extrahelical depending on temperature, while the pyrimidine bases stack poorly and are likely to be extrahelical (13).
Recent studies in eucaryotes, however, suggest that the A-rule may not
be a universal mechanism for preferential incorporation of A opposite
noncoding template lesions. Sarasin and co-workers (14, 15) using a
shuttle vector system, replicated in monkey COS7 cells, observed an
essentially random incorporation of nucleotides opposite abasic
lesions. In a study of mutagenesis at apurinic sites in normal and
ataxia telangiectasia human lymphoblastoid cells, Drinkwater and
co-workers (16) found that incorporation of G was favored. Kunz, Demple
and co-workers (17), using a yeast plasmid system that mimics
chromosome behavior, found that "chromosomal" mutations in
Saccharomyces cerevisiae were not in accordance with the
A-rule, also showing a preference for G incorporation. Ohtsuka and
co-workers (18) observed activation of a c-Ha-ras gene by an
abasic site-induced point mutation having preferential incorporation of
T opposite the lesion. A second ras-activating point
mutation was located at the 5-site adjacent to the abasic lesion (18).
Remarkably, in a shuttle vector system in yeast cells, Gibbs and
Lawrence discovered a "C-rule" where incorporation of C occurred
opposite a site-directed abasic lesion 80% of the time (19). In the
light of an accumulating body of evidence indicating significant
differences in the processing of abasic lesions in procaryotic and
eucaryotic cells, it is important to measure nucleotide incorporation
specificities for eucaryotic DNA polymerases, especially the eucaryotic
repair enzyme pol
, which has no known procaryotic counterpart.
In this paper, we use human pol and pol
to measure
deoxyribonucleotide incorporation and extension efficiencies at a
site-directed tetrahydrofuran abasic analog. The incorporation
efficiencies were measured in the presence of different upstream and
downstream nucleotides adjacent to the template abasic lesion. We
distinguish between two types of incorporation mechanisms: "direct"
incorporation and a novel form of "misalignment" incorporation.
Direct incorporation refers to nucleotide insertions occurring directly
opposite a lesion, and misalignment incorporation is defined by
nucleotide insertions occurring opposite a normal template base
downstream from the lesion, on a misaligned primer-template terminus.
The novel form of misalignment incorporation, carried out by pol
, does not conform to misaligned p/t DNA structures reported previously (20-22). We use the term "dNTP-stabilized misalignment" to refer to our model for pol
-catalyzed misalignment incorporation at abasic
lesions, which will be distinguished from a "standard misalignment" model presented previously (20-22). We are concerned primarily with
two issues germane to translesion synthesis. First, we address the
degree to which pol
and pol
incorporate nucleotides via direct
incorporation in compliance with the A-rule. Second, we determine the
extent to which the polymerases utilize direct and misalignment
synthetic mechanisms to catalyze lesion bypass.
Materials
EnzymesHuman pol was purified as described (23). The
turnover number for pol
is from 0.5-0.8 nucleotides
s
1 when assayed using poly(dA)-oligo(dT) at 20 °C, pH
7.4. Human pol
(2000 units/mg, see Ref. 24), a generous gift of Dr.
T. S. F. Wang, refers to the large catalytic subunit of holoenzyme and
was purified as described (24). Both highly purified enzyme preparations were free of detectable 3
-exonuclease activities. 1 DNA
polymerase unit is defined as the incorporation of 1 nmol of dNMP into
DNA per h at 37 °C. T4 polynucleotide kinase was purchased from U. S. Biochemicals.
Nonradioactive nucleotides were purchased from
Pharmacia Biotech Inc. [-32P]ATP (4000 Ci/mmol) was
purchased from ICN Radiochemicals.
Abasic
(1,4-anhydro-2-deoxy-D-ribitol) phosphoramidite was
synthesized as described previously (25). 42-mer templates containing a
single abasic site and control templates with no abasic site were
synthesized on an Applied Biosystems 392 DNA/RNA synthesizer, as were
the different primers. Oligodeoxyribonucleotides with a 5-phosphate
group used to form gaps for pol
were synthesized by Lynn Williams
at the University of Southern California Comprehensive Cancer
Center.
For studies of insertion opposite an abasic
site by pol , a 5-nucleotide gap with an abasic site in its center
was formed by annealing a 42-mer template, 5
-TTC TAG GTT TGC TAA CAT
ACT TCN XMA TAA GGA GTC TTA ATC-3
, where the X
designates the tetrahydrofuran abasic lesion; N and M are,
respectively, the downstream and upstream nearest-neighbor bases to be
varied (Fig. 1, a and b). The upstream
oligonucleotide primer was 5
-GAT TAA GAC TCC TTA-3
, and the
downstream oligonucleotides used to form a 5-nucleotide gap for pol
was 5
-AAG TAT GTT AGC AAA CCT AGA A-3
. The downstream
oligodeoxyribonucleotide was phosphorylated at its 5
-end (Fig. 1). The
5
-phosphate group on the downstream oligonucleotide and a gap size of
fewer than six nucleotides are necessary for processive behavior by pol
(26). The same studies with pol
used no downstream
oligonucleotide; pol
activity was inhibited by approximately 2-fold
in a short gap filling reaction (data not shown). For studies of
misaligned versus direct extension, the same templates were
used but with upstream primers that were three nucleotides longer and
covered the abasic site with A, C, G, or T. These templates were also
annealed to a downstream oligonucleotide one nucleotide shorter than
the one in the insertion assays without the first 5
-A, to form a
3-nucleotide gap (Fig. 1c). All primers and templates were
purified by polyacrylamide gel electrophoresis.
Methods
Conditions for Determining Kinetic Constants5-End
labeling of primers with 32P was carried out using
T4-polynucleotide kinase in a reaction containing 56 mM
Tris-HCl, pH 7.7, 7 mM MgCl2, 13 mM
dithiothreitol, 18.5 nM DNA, 0.4 µM [
-32P]ATP (4000 Ci/mmol), and T4-polynucleotide kinase
(3 units/97-µl reaction). Reactions were incubated at 37 °C for 60 min and terminated by boiling at 100 °C for 5 min. Annealing
reactions for pol
were carried out by incubating 7.5 nM
primer, 7.5 nM template, 38 nM downstream
oligonucleotide in pol
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, 2%
glycerol) at 37 °C for 1 h followed by a gradual cooling to
room temperature. For pol
, p/t DNA was annealed by incubating 7.5 nM primer with 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, 1 mM
-mercaptoethanol) at 37 °C for 1 h, followed by a gradual
cooling to room temperature.
Prior to carrying out kinetics experiments, time courses
were run to determine conditions for measuring insertion and extension efficiencies at abasic template sites. Reactions were performed by
mixing equal volumes (5 µl) of annealed primer-template and deoxyribonucleotide in reaction buffer (see annealing conditions). The
final concentration of primer-template in all reactions was 3.75 nM. Pol (0.2 units) or pol
(0.2 units) were added
and the reactions were incubated at 37 °C for 30 min.
Four p/t DNA constructs were used to measure incorporation opposite the model abasic (tetrahydrofuran) lesion. Although incubation times of about 2 min were sufficient to measure incorporation opposite the lesion for many of the mispairs, there remained a number of sequence contexts in which incorporation could not be detected, or where there was only slight incorporation or extension at or beyond the lesion. We found it necessary to use 30-min incubation times to detect incorporation bands for each of the templates over the range of dNTP concentrations used for the kinetic measurements. We estimate that about half of the template-primers have been extended during the 30-min incubation so that approximately 35% of the p/t DNA encounter a polymerase once, 10% twice, and 5% three times. Since there was no significant difference in the fraction of primers utilized during each experiment, the relative incorporation and extension efficiencies are not biased by the occurrence of multiple polymerase-p/t DNA encounters (27).
For each reaction, the dNTP substrate for incorporation opposite X was varied in concentration over a range of 10 µM to 1.5 mM. For the time courses, the substrate concentration used for incorporation opposite the lesion was held constant at 1 mM. The reactions were carried out under running start conditions and contained from 5 to 50 µM dTTP for incorporation opposite two dAMPs upstream of the lesion. The dTTP concentrations were chosen to extend the primers as rapidly as possible subject to the constraint that no measurable dTMP was incorporated opposite the lesion itself. Reactions were terminated by adding 2 volumes of 20 mM EDTA in 95% formamide to chelate Mg2+ prior to heating, heating to 100 °C for 5 min, and placing the reactions on ice for about 5 min. The samples were then loaded on an 18% polyacrylamide gel and the extended single-stranded DNA primers were separated according to length by electrophoresis (9, 28).
A gel fidelity analysis was used to determine the kinetics of
incorporation of each of the four deoxynucleotides opposite the lesion
as a function of deoxynucleotide concentration (9, 28). Integrated
polyacrylamide gel band intensities were measured on a PhosphorImager
using Imagequant software (Molecular Dynamics, Sunnyvale, CA). The
nucleotide incorporation efficiency (apparent Vmax/Km) opposite a lesion,
measured in a running-start reaction, can be obtained by measuring
IX/IX-1, where IX is the integrated
gel band intensity of a primer extended to the site opposite the lesion
and beyond, and IX-1 is the integrated gel band intensity
of a primer extended to a site just prior to the lesion (9, 28). A plot
of IX/IX-1 as a function of the target dNTP
substrate concentration results in a rectangular hyperbola whose slope
in the linear region is given by apparent
Vmax/Km. Apparent
Km and relative Vmax values
were obtained using a nonlinear least squares fit to the rectangular
hyperbola. In p/t DNA sequence contexts where incorporation and
extension were relatively inefficient, plots of
IX/IX-1 versus dNTP concentration showed little or no curvature; in these cases, apparent
Vmax/Km values were obtained
by a least squares fit of the data to a straight line. The same
analysis was also used to calculate apparent kinetic constants for
extension beyond an abasic site (following subsection). Based on a
series of time course measurements, kinetic data were obtained using
30-min incubations. The data for both enzymes were in the linear range
for incorporation and extension at the abasic lesions for each of the
p/t DNA constructs, with the possible exception of dAMP incorporation
opposite X by pol . We note, however, that the relative
efficiencies of incorporation of dAMP compared to dGMP opposite
X are consistent with previous measurements using pol
to
copy a tetrahydrofuran moiety (9, 10).
Reactions were carried out as
described above except that the abasic site was covered by the upstream
primer with A, C, G, or T (see, e.g. Fig. 1c).
These reactions were carried out using standing-start conditions. The
reactions were run in the presence of only one nucleotide, either the
one complementary to the first base downstream of X or to
the second nucleotide downstream of the lesion. The concentration of
dNTPs was varied between 10 and 500 µM, and the reactions
were run for 30 min. The extension efficiency,
Vmax/Km, is proportional to
(IX+1/Io) × 1/t, where
IX+1 is the integrated band intensity for band(s) extended
beyond the abasic lesion, Io is the integrated band
intensity of the unextended primer band located opposite the lesion,
and t is the reaction time. The proportionality constant is
the same for incorporation of A, T, G, or C opposite X in
all sequence contexts (29) so that a comparison of the ratio
IX/IX-1 for each nucleotide provides a direct
measure of the relative extension efficiencies. In cases where
Io is not IX+1, then Io is replaced in the equation for fext by
Io + 0.5 IX+1 to correct for the change in
concentration of input p/t DNA during the course of the reaction
(30).
Apparent equilibrium dissociation constants were determined
for binding of pol or pol
to the p/t DNA used in the extension assays as described in Ref. 31.
To investigate the specificity of nucleotide incorporation
opposite abasic template sites using eucaryotic pol and pol
, template DNAs were synthesized with a site-directed model
(tetrahydrofuran) abasic lesion (25) placed adjacent to each of the
four different upstream and downstream nearest-neighbor bases (Fig.
1). The abasic lesion is denoted by the symbol
X. Steady-state kinetic measurements were performed to
determine nucleotide incorporation efficiencies opposite X
(9, 32) and efficiencies of extension downstream from X (30,
32, 33).
Nucleotide
incorporation kinetics for pol and pol
were measured in
running-start reactions, by varying the downstream base adjacent to the
lesion. In the running-start reaction, two Ts are incorporated prior to
encountering an abasic lesion, followed by incorporation of dNMPs A, C,
G, or T, at different concentrations, opposite the lesion,
X. Representative kinetic data are presented in which G is
the base immediately downstream from the lesion (Fig.
2). The data illustrate that pol
incorporated all four nucleotides opposite X, favoring misalignment
incorporation of dCMP which is complementary to the template base to
the 5
-side of the lesion (Fig. 2a, dCTP lanes). In
contrast, pol
strongly favored incorporation of dAMP and to a
lesser extent dGMP (Fig. 2b). Pol
also catalyzed
incorporation of dCMP, but with exceedingly low efficiency (Fig.
2b, dCTP lanes).
Two distinct mechanisms are responsible for the incorporation of C in
this p/t configuration: (i) direct incorporation of dCMP opposite
X and (ii) misalignment incorporation of dCMP opposite the
downstream template G. Note that misalignment synthesis, where the
incoming dNTP stabilizes a misaligned p/t structure, has not been
reported previously. We refer to this novel type of synthesis as
"dNTP-stabilized" misalignment synthesis to distinguish it from the
standard "Streisinger" misalignment synthesis (20, 21, 34, 35) in
which the p/t DNA forms a transiently misaligned structure (Fig.
3).
Time courses, covering a range from 2 to 60 min, were performed to
measure nucleotide incorporation opposite X with the same
p/t sequence used for the kinetic analysis shown in Fig. 2 (data not
shown). The time course data were consistent with the kinetic data,
i.e. the same polymerase-specific differences in the
abilities of the two human polymerases to incorporate nucleotides opposite an abasic lesion were observed at each reaction time point.
Pol behaved in accordance with the A-rule since it readily incorporated dAMP, and less readily dGMP, opposite the lesion, whereas
incorporation of pyrimidines opposite X was barely detectable, even following a lengthy (60 min) reaction period (data not
shown). Pol
did not follow the A-rule since it incorporated all
four dNMPs opposite X in a relatively impartial manner at
each time point (data not shown).
Analysis of the data for each of the four downstream nearest-neighbors
demonstrates pol -catalyzed incorporations were most efficient when
the downstream template base was complementary to the incoming dNTP
substrate (Fig. 4a,
Vmax/Km values indicated by
arrows pointing downward along the diagonal). The large
apparent Vmax/Km values along
the diagonal suggest that a major fraction of misincorporations taking
place opposite the lesion are actually "correct" incorporations in
which an incoming dNTP is positioned opposite its complementary
template base immediately downstream from the lesion. Except in the
case of a downstream template A, misincorporations by pol
are
dominated by this mechanism. Nucleotide misincorporations caused by p/t
misalignments, as documented by Kunkel and co-workers (20, 21), contain
a "looped out," i.e. extrahelical base (in this case a
looped out abasic moiety) stabilized by H-bonded base pairs surrounding
the looped out moiety (see, e.g. Fig. 3, standard
misalignment structure). However, the misalignment incorporations found
for pol
in running-start reactions (Fig. 4a) are novel
because, there is no base pair available downstream from the lesion to
stabilize a putatively looped out X (Fig. 3, dNTP-stabilized
misalignment structure). Instead, stabilization of the misaligned
structure occurs when a dNTP complementary to the template base
downstream from X binds in the active site of pol
.
The incorporation pattern for pol (Fig. 4b) differs from
that of pol
. The largest apparent
Vmax/Km values for pol
correspond to incorporations of dAMP opposite X. Incorporations of dGMP opposite X were 5-12-fold lower in
efficiency compared to dAMP. Incorporation of pyrimidine nucleotides opposite X occurred with lowest efficiencies. This trend is
in accordance with A-rule behavior (8) for incorporation at abasic
lesions in vitro (3, 9, 10). The lack of correlation between
the magnitudes of Vmax/Km and
the presence of a template base downstream from the lesion
complementary to an incoming dNTP suggests that pol
acts
principally by incorporating nucleotides directly opposite
X, and not by dNTP-stabilized misalignment.
The 12 "off-diagonal"
Vmax/Km values for pol are dominated by direct incorporations occurring opposite the lesion (values that do not fall on the diagonal designated by the
arrows, Fig. 4a). A synopsis of the 12 direct
incorporation Vmax/Km values
suggests that pol
incorporates nucleotides in a less biased manner,
in comparison to pol
which favors incorporation of dAMP, and to a
lesser extent dGMP. We conclude that, in relation to the incorporation
of dAMP, pol
incorporates pyrimidine nucleotides in a more unbiased
manner than pol
. The data further suggest that, when copying
identical sequence contexts in the direct incorporation mode, pol
may incorporate pyrimidine deoxynucleotides with higher absolute
efficiencies (Vmax/Km values)
than pol
(Fig. 4), but this point has not been conclusively
demonstrated.
The presence of an abasic lesion caused a strong reduction in
incorporation efficiencies for both polymerases, typically by factors
of between 104- and 105-fold compared to normal
template sites. Vmax/Km
values for dNMP incorporation at normal template sites (where
X is replaced by a normal template base) were approximately
105 M1 to 106
M
1 using pol
on gapped p/t DNA; pol
activity on ungapped p/t DNA was very low (data not shown).
Corresponding measurements with pol
resulted in
Vmax/Km values on the order
of 105 M
1, with activities
roughly 2-fold higher on ungapped DNA (data not shown). The reduction
in incorporation and extension efficiencies at abasic template sites
compared to normal sites reflects a strong kinetic block,
i.e. an impediment in synthesis where reduction is not
caused by the inability of pol
to bind in the vicinity of
X; we found that the apparent equilibrium binding constants for both enzymes were not altered significantly in the presence of an
abasic lesion (data not shown).
Nucleotide extension kinetics
for pol and pol
were measured in a standing-start reaction by
varying the concentration of dNTP substrates targeted for incorporation
opposite a complementary template base either one base downstream from
the lesion site, corresponding to direct extension, or two bases
downstream from X, corresponding to misalignment extension,
in accordance with a standard misalignment structure (Fig. 3 and Fig.
5). Note that in the extension reaction, the
primer-3
-terminal base covers the lesion (Fig. 1c).
Pol extends past an abasic lesion using misaligned primer termini
by incorporating the dNMP complementary to the template site
two bases downstream from X (Fig. 5, a
and b, right-hand gels). Continued synthesis on the
misaligned p/t DNA would lead to a
1 nt deletion. Tandem base
substitutions could also occur if continued synthesis were to take
place from a realigned primer-template. In the relatively unlikely
event that template realignment had occurred prior to continued
synthesis, then following realignment of the template, dGMP which had
been "correctly" incorporated opposite C, two bases downstream from
X, would then be relocated opposite G to form a dCMP·X, dGMP·G
tandem mispair (Fig. 5a), or opposite T to form a dAMP·X,
dGMP·T tandom mispair (Fig. 5b).
A minute amount of direct extension onto a correctly aligned primer
terminating in C opposite X was observed in the lane
corresponding to 500 µM dCTP (Fig. 5a, left-hand
gel); a small amount of direct extension occurred on a correctly
aligned primer terminating in A opposite X (Fig. 5b,
left-hand gel). In the case of pol , primers terminating in A
were extended with high efficiency both by direct and standard
misalignment modes of synthesis, whereas primers terminating in C were
extended poorly using a misaligned primer and were not measurably
extended on a correctly aligned primer.
A synopsis of extension efficiencies for direct and standard
misalignment synthetic modes is shown in Fig. 6.
Extensions past abasic lesions for pol occurred almost exclusively
from misaligned primer termini (Fig. 6a). Misalignment
extension for pol
was strongly favored from dAMP·X and
dGMP·X primer termini (Fig. 6b). An important
additional feature of pol
extension is that efficient direct
extension also occurred for primers terminating in A while less
efficient direct extension took place from primers terminating in G. Note that three of the four template strands contained CT at the 5
-end
of the 3-nucleotide gap (Fig. 6). The template having a primer
terminating with G opposite X has the CT replaced by AT to
avoid generating both one and two base loops in which the primer-3
-G
pairs with a template C adjacent to the lesion or a second C two bases
downstream from X. We have observed that pol
extends
transiently misaligned primers containing putative two-base loop-outs
(data not shown).
Nucleotide Incorporation Efficiencies Opposite X Measured as a Function of Upstream Sequence Context
Nucleotide incorporation
efficiencies were measured in a running-start reaction on templates
with a different upstream template base present at the 3-side of
X. The downstream template base (to the 5
-side of
X) was held constant (Fig. 7). Pol
favored incorporation of dGMP opposite the template C downstream from
the lesion in the four p/t configurations, via dNTP-stabilized misalignment incorporation (Fig. 7a). As expected, pol
strongly favored incorporation of dAMP opposite X (Fig.
7b). Data were obtained for the template 3
- ...
AAXCC.. (Fig. 7) independently from that shown in Fig. 4
(second panel from the top); the two data sets are in good
agreement.
For pol , an unexpected finding was the remarkably strong effects
caused by changing just one upstream base. Incorporation opposite
X occurred much more efficiently on the template 3
- ... AGXCC ... compared to 3
- ...
ATXCC; the incorporation efficiencies were greater by
factors of 51-fold, >66-fold, 12-fold, and 1.6-fold for A, C, G, and T
opposite X (Fig. 7a, third and
fourth panels). The corresponding comparisons for pol
,
while less dramatic, still demonstrate a significant influence of the upstream base pair on incorporation opposite the lesion (Fig. 7b, third and fourth panels). Large
upstream sequence effects were observed for pol
when comparing
templates 3
- ... ACXCC ... and 3
...
ATXCC (Fig. 7b, second and
fourth panels); here the incorporation efficiencies were
greater with ACXCC by factors of 10-fold, 9-fold, 16-fold,
and 2-fold for A, C, G, and T, respectively. We verified that there
were no significant differences in primer utilization for any of the
DNAs, ruling out the possibility that the large upstream
nearest-neighbor effects on incorporation opposite the lesion could be
caused by differential utilization of the four p/t DNAs (data not
shown).
Pol failed to incorporate the next correct nucleotide, dCMP opposite G
(adjacent to X), when the 3
-primer terminus was placed
directly opposite the abasic lesion (Fig. 5a, left-hand gel). In contrast, pol
was able to incorporate dCMP readily opposite the same neighboring template G in a running-start reaction (Fig. 2a, dCTP lanes). Thus, there appears to be a
significant difference in lesion bypass efficiency when pol
initially incorporates a nucleotide opposite X in contrast
to extension from a pre-existing primer terminating opposite
X. We have made similar observations using other p/t DNA
configurations (data not shown).
An analysis of product lengths in the gap-filling reaction
for pol reveals that one base deletions and base substitutions can
result from lesion bypass caused by dNTP-stabilized misalignment. Extension of 32P-labeled primer bands by addition of 1-5
nt were observed as discrete gel bands when pol
copied the gapped
templates, 3
-AAXGC (Fig. 8a) and
3
-AAXCC (Fig. 8b). Lesion bypass occurred when
either 4 or 5 nt were added during the extension reaction. The addition
of 4 nt on a misaligned template (X out of the helical
plane) would result in a 1-nt deletion, while the addition of 5 nt on a
realigned template (X in the helical plane) would result in
a base substitution.
Doublet bands were observed at the template G position when dGTP and
dCTP were present in the reaction (Fig. 8a, lanes 2-4, lower two
arrows). The less intense lower band of the doublet migrated in
the same position as a primer band extended in the presence of dCTP but
in the absence of dGTP (Fig. 8a, lane 1); thus, the lower
doublet band (Fig. 8, lower arrow) corresponds to a primer
extended by incorporation of 4 nt, with dCMP at its 3 terminus. The
intense upper doublet band (Fig. 8, middle arrow) corresponds to addition of 4 nt onto the primer, with dGMP at its 3
terminus. Primers were also extended by incorporation of 5 nt as shown
by the presence of a low intensity band opposite C, migrating above the
doublet (Fig. 8a, lanes 2-4 and 6, upper arrow).
The origin of the primer extension bands for the 3-AAXGC
template can be inferred from the dNTP-stabililized misalignment model
(Fig. 3). Formation of the relatively intense upper doublet band (Fig.
8, middle arrow) is likely to have occurred by incorporation of dCMP opposite G on a misaligned template (X looped out of
the helix) followed by incorporation of dGMP opposite C using the same
misaligned template. We suggest that the p/t structure responsible for
generating the upper band would lead to a
1 nt deletion. This
structure is stabilized by the presence of C:G and G:C base pairs
downstream from the putative looped-out abasic moiety.
The lower doublet band (Fig. 8, lower arrow) was probably
formed by the following steps: (i) incorporation dCMP opposite G on a
misaligned template, (ii) realignment of the template strand (placing
dCMP opposite X), (iii) incorporation of a second dCMP
opposite G, (iv) dissociation of pol , leaving a partially filled
gap. The upper band of the doublet is much more intense than the lower
band. This difference in intensities is consistent with the idea that a
misaligned structure containing consecutive C:G and G:C base pairs,
downstream from the looped-out lesion, should be considerably more
stable than a realigned structure containing C opposite X
followed by a single C:G base pair. NMR measurements showed that both C
and X were located extrahelically when they were located
opposite one another in double-stranded DNA (13).
The band running above the doublet opposite C (Fig. 8, upper
arrow) corresponds to extension of the primer by 5 nt, to
completely fill the gap. Therefore, template realignment can occur,
allowing incorporation of dGMP opposite C, in spite of the reduced
stability of the realigned structure relative to the misaligned
structure. The presence of a band opposite C implies that base
substitutions can result from dNTP-stabilized misalignment, although,
they would be much less likely to occur than 1 base deletions (Fig.
8, middle arrow).
Further insight into dNTP-stabilized misalignment pathways can be arrived at by investigating the dependence of band intensities on dGTP concentration. As dGTP is increased relative to dCTP, the lower doublet band decreases in intensity (Fig. 8a, lanes 2-5). Incorporation of dCMP occurs efficiently opposite G with X out of the plane of the helix. As the concentration of dGTP is increased relative to dCTP, it becomes much easier to incorporate dGMP opposite C, from a misaligned template, than to incorporate a second dCMP opposite G from a realigned template. In other words, at high dGTP/dCTP ratios the template does not appear to have sufficient time to realign prior to incorporation of dGMP opposite C.
There is also a marked decrease in intensity of the band opposite C
accompanying an increase in dGTP concentration (Fig. 8a, lanes
4 and 5, upper arrow), suggesting that a 1 base
deletion occurs in preference to a base substitution. At high dGTP/dCTP ratios, the addition of two complementary nucleotides, dCMP opposite G
followed by dGMP opposite C, acts to stabilize the misaligned template,
favoring
1 base deletions. At low dGTP/dCTP ratios, base
substitutions can occur because the template has sufficient time to
realign. Further evidence supporting the occurrence of template
realignment is the presence of a band opposite C when the reaction is
carried out initially with dTTP (running-start nucleotide) and dCTP
followed, at 10 min, by the addition of dGTP (Fig. 8a, lane
6). Following dCMP incorporation opposite G on a misaligned
template, the template realigns in the absence of dGTP and
incorporation of dCMP opposite G can again occur (see, e.g.
Fig. 8a, lane 1). When dGTP is added 10 min later,
incorporation of dGMP opposite C occurs on the realigned template (Fig.
8a, lane 6), leading to a base substitution.
An analysis of primer extension products using a 3-AAXCC
template demonstrates that template realignment can occur even if the
misaligned structure is stabilized by two downstream G:C base pairs
(Fig. 8b, lanes 1 and 2). Primer extension
corresponding to the addition of dGMP opposite C (with X out
of the helical plane), followed by addition of dGMP opposite C on the
misaligned template, gives rise to a
1 base frameshift at low dGTP
(100 µM) concentrations (Fig. 8b, lane 1). At
a higher dGTP concentration (600 µM), both base
substitutions and
1 frameshifts occur (Fig. 8b, lane 2).
The ability to fill the 5-nt gap at high dGTP concentration suggests
that the template can undergo realignment (placing C opposite
X), even though template realignment is far less favorable
than retention of the misaligned template structure having two G:C base
pairs downstream from a looped-out X. The ability to fill in
the 5-nt gap completely, at a high dGTP concentration (Fig. 8b,
lane 2), suggests that base substitutions can result even though
template realignment is a kinetically unfavorable process.
The data also show that pol is processive in synthesizing past the
lesion provided that the misaligned template does not realign. Pol
tends to dissociate following template realignment; a discrete
extension band is observed opposite X (Fig. 8a, lanes
2-4). However, the band corresponding to insertion opposite X is absent (Fig. 8a, lanes 5 and 6)
when the template is misaligned; pol
does not dissociate from the
template when adding dCMP opposite G followed by dGMP opposite C in the
dNTP-stabilized misalignment mode. Note, however, that two barely
detectable doublet bands can be seen opposite X in
lanes 5 and 6. These bands arise from inefficient
incorporation of either dGMP or dCMP directly opposite the
lesion, after which pol
dissociates.
It has been proposed that the A-rule (8), defined as the preferential incorporation of A at abasic DNA template lesions, serves as a DNA polymerase default mechanism allowing bypass of noncoding lesions encountered at a replication fork. Previous studies showed that polymerases purified from eucaryotic and procaryotic cells behave in accordance with the A-rule, preferentially incorporating A opposite abasic lesions with about a factor of 10 higher efficiency than G and with about a 50-fold greater efficiency than either C or T (9, 10).
However, recent in vivo experiments suggest that the A-rule
may not be operative at abasic sites in eucaryotes. Data from eucaryotic systems demonstrate that incorporation can be random (14,
15), or that G (16, 17), T (18), and C (19) can be favored, depending
on the particular system studied. The eucaryotic results raise the
interesting possibility that perhaps one or more eucaryotic polymerases
exhibit a broad specificity range for incorporation at abasic sites. We
have tested this possibility by measuring the efficiency of nucleotide
incorporation at abasic lesion sites using pol , a repair polymerase
found only in eucaryotic organisms.
Kinetic determinations of nucleotide incorporation efficiencies were
performed at site-directed abasic lesions (X) placed next to
four different downstream template bases, for each of the four dNTP
substrates (Fig. 4). Nucleotide incorporation takes place directly
opposite X in 12 of the 16 dNTP-p/t configurations. In the
four configurations, where a dNTP substrate is complementary to a
template base immediately downstream from the lesion, incorporation can
occur either directly opposite X on a properly aligned
primer, or opposite a downstream template base on a misaligned primer.
Side-by-side comparisons were made for pol and pol
using
identical p/t DNA constructs.
Two definitive differences in the properties of pol and pol
were found for nucleotide incorporation at abasic lesions. First, pol
does not exhibit A-rule behavior while pol
does (Figs. 2 and
4). Second, pol
incorporates nucleotides with higher efficiency,
using misaligned primer-3
-termini (data indicated by arrows
along the diagonal, Fig. 4a), while pol
does not (Fig. 4b). The misalignment mechanism for pol
is different
from that previously observed (20-22, 36, 37) because it does not
require the presence of H-bonded structures surrounding the
extrahelical moiety as postulated for the standard misalignment model,
shown in Fig. 3. Instead, the presence of an incoming dNTP
complementary to the template base downstream from the lesion is
sufficient to cause misalignment incorporation to take place; we refer
to this novel type of misincorporation as "dNTP-stabilized
misalignment" incorporation (Fig. 3). We have also shown that this
novel mode of misalignment incorporation for pol
favors the
occurrence of
1 frameshifts but can also give rise to base
substitutions (Fig. 8). It has previously been shown that pol
catalyzes
1 frameshifts and base substitutions by the standard
Streisinger slippage mechanism (20).
The 12 apparent
Vmax/Km values off the
diagonal correspond to pol incorporations occurring directly
opposite the lesion, i.e. not via p/t DNA misalignment
synthesis (Fig. 4a). With A downstream from X,
direct incorporation of G opposite X was favored slightly
over A and C; with C downstream, A was favored by roughly 7-fold over C
and T; with G downstream, A and T were almost equivalent and favored by
about 2-fold over G; with T downstream, C and G are roughly equivalent
and favored by about 2-fold over T. Thus, pol
appears to operate in
a robust and less than partial manner than pol
with respect to
direct incorporation opposite abasic template lesions. The properties of pol
are obviously different because A was favored over G for
incorporation opposite X by factors ranging from 5-12-fold, for all p/t configurations, while incorporation of the pyrimidine nucleotides was far less efficient.
A molecular basis to rationalize the A-rule stems from NMR studies
showing that A is stacked within the plane of the DNA helix (11-13), G
is intrahelical at low temperatures but is melted prior to denaturation
of the helix, and that C and probably T are extrahelical (13). It is
possible that base stacking interactions between incoming dNTPs and
primer-3-termini differ significantly for pol
in relation to other
polymerases that have been investigated, e.g. pol
, human
immunodeficiency virus-1 and avian myeloblastosis virus reverse
transcriptase (32). The properties of pol
concerning untemplated
incorporation at abasic template sites on physiologically relevant p/t
configurations used here are also in contrast to the A-rule-like
results observed for untemplated end-addition reactions, noted both in
solution (38) and in crystals of enzyme and blunt-ended DNA (39). In
the end-addition reactions, base stacking interactions for the primer
base and incoming base may be more important for stabilizing the
transition state intermediate than in the case of abasic site
untemplated synthesis on a primer-template. In the latter case, pol
-DNA interactions may impose a bend in the primer-template such that
stacking between the primer base and incoming base is not critical to
the free energy of stabilization of the transition state (40).
In a standing-start
reaction, pol appeared to have great difficulty adding a next
correct dNTP onto pre-existing primers terminating opposite an abasic
lesion (Fig. 5a). In contrast, in a running-start reaction,
the enzyme had essentially no difficulty carrying out translesion
synthesis by first incorporating a nucleotide using a misaligned
primer-template and then using a realigned template to continue
synthesis past the lesion by direct addition of the next correct dNMP
(Fig. 2a, dCTP lanes). Although we have not attempted to
carry out a systematic investigation using a large number of p/t DNA
sequences, this observation does not appear to be restricted to
specific sequence contexts.
Conceivably, pol may assume different conformations depending on
whether it binds at a pre-existing primer terminus opposite a lesion or
is bound at the identical site just after having inserted a nucleotide
opposite X. The difficulty of pol
to extend a
pre-existing primer by addition of a dNMP complementary to the template
base immediately downstream from the lesion, even after a 30-min
incubation (Figs. 5, a and b, left-hand gels),
suggests that the enzyme is inhibited in forming a kinetically
competent complex when bound opposite a lesion on a properly aligned
primer terminus. However, when pol
was bound to a misaligned primer (indicated in Fig. 5, a and b, by the looped-out
X shown out of the helical plane, right-hand
gels), the enzyme was easily able to add the next correct dNMP
two bases downstream from the lesion. The underlying reasons
for these interesting kinetic differences are not known and merit
further study. These marked differences in standing-start compared to
running-start kinetics can be exploited in delineating rate-limiting
steps in the nucleotide incorporation pathway for pol
.
Another interesting aspect of the data shown in Fig. 5 is that pol extends past an abasic lesion using misaligned primer termini by
incorporating the dNMP complementary to the template site
two bases downstream from X (Fig. 5, a and
b, right-hand gels). Following realignment of the template,
dGMP which had been correctly incorporated opposite C, two bases
downstream from X, is then relocated opposite G to form a
dGMP·G mispair (Fig. 5a), or opposite T to form a dGMP·T
mispair (Fig. 5b). This mechanism, strongly favored by pol
may explain the interesting observation of Kamiya et al.
(18) that c-Ha-ras gene mutations occur both at the site of
an abasic lesion and at an adjacent downstream site.
We expected that
differences in the efficiencies of nucleotide incorporation opposite
X would depend on the identity of the base pair preceding
the lesion and on the specific polymerase used in the assay. Sequence
context effects on fidelity at normal and aberrant template sites have
been well documented, and models have been proposed to address how
polymerase parameters might modulate the appearance of nucleotide
incorporation hot and cold spots (41-43). However, we did not expect
to observe the extremely large effects exerted by upstream base pairs
on incorporation efficiencies opposite X (Fig. 7). For
example, when T was replaced by G as the neighboring upstream template
base, pol incorporated A and C opposite X with 51-fold
and >66-fold higher efficiencies, respectively (Fig. 7a,
third and fourth panels). Large upstream
nearest-neighbor effects, on the order of 10-15-fold, were also
observed for pol
(Fig. 7b, second and
fourth panels).
The ability of pol
to incorporate nucleotides in a relatively indiscriminant manner
opposite an abasic lesion in the direct incorporation mode (Fig.
4a), and opposite a base downstream from an abasic lesion in
the dNTP-stabilized mode, suggests a possible resolution for the A-rule
disparity between procaryotic and eucaryotic organisms. However, an
important cautionary note is that although a requirement for pol
has been shown in short gap filling DNA repair synthesis pathways (44,
45), there is no evidence showing that pol
actually carries out
translesion synthesis during in vivo replication or repair.
Nevertheless, one can speculate that replication complexes involving
pol
and pol
stall when encountering an abasic lesion during
chromosomal replication. Dissociation of the replication complex could
allow pol
access to carry out error-prone translesion synthesis.
Then, following dissociation of pol
, reassociation of the
replication complex could take place at a nascent primer terminus
downstream from the lesion. It is perhaps even more likely that pol
might act during excision repair to copy an abasic site located within
a repair gap created by a targeted removal of a UV-induced lesion on
the opposite strand.
Replication and repair polymerases appear to have overlapping cellular
roles; for example, pol has been implicated in base excision repair
in yeast (46) and repair of human fibroblast DNA (47). Although it
remains to be determined if either pol
or pol
exhibit broad
nucleotide incorporation specificities at abasic lesions, proofreading
exonucleases are known to strongly inhibit abasic lesion bypass
in vitro (29). In contrast to both pols
and
which
contain active proofreading activities (48), pol
has no detectable
proofreading activity (48, 49).
The presence of an abasic lesion results in a strong reduction in
incorporation efficiencies, on the order of
104-105-fold (data not shown; see also Ref. 9).
The reduction in incorporation and extension (32) efficiencies at
abasic template sites compared to normal sites reflects a strong block
to DNA synthesis in the vicinity of an abasic lesion. Therefore, it is
likely that other proteins might interact with polymerases at the site
of an abasic lesion to stimulate translesion synthesis and possibly to
alter nucleotide insertion specificities. The SOS-induced protein
complex, UmuDC, is required in vivo to observe error-prone
translesion bypass in E. coli, see e.g. Ref. 50.
A recent result supporting the possibility that polymerase
specificities may depend on interactions with lesion bypass accessory
proteins is that two analogs of umuD
C, mucAB and rumAB, were shown to alter the relative
frequencies of mutations at cyclobutane dimers in vivo (51).
However, there is as yet no evidence for the presence of UmuD
C-like
translesion bypass proteins in eucaryotic cells. The yeast REV 1 protein has recently been shown to catalyze incorporation of C opposite
abasic lesions in vitro (52). Perhaps this activity is
responsible for the 80% level of dCMP incorporation opposite
X observed on a shuttle vector in yeast (19). However, the
deoxycytidyl transferase activity of REV1 does not explain the favored
incorporation of G at abasic lesions in the yeast chromosomal model
system (17).
When allowance for each of these caveats is made, the salient point
remains that pol is the first example of an enzyme able to copy
abasic lesions in a generally impartial manner. We suggest that the
relaxed incorporation specificity of pol
provides a plausible
biological mechanism to account for the lack of an A-rule in
eucaryotes.