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INTRODUCTION |
Abasic (apurinic/apyrimidinic;
AP)1 sites represent one of
the most frequently formed DNA lesions in eukaryotic cells. Base loss
can occur by spontaneous hydrolysis of the N-glycosylic bond or by the action of DNA glycosylases on damaged bases. It has been
estimated that a mammalian cell loses up to 10,000 purines/day from its
genome (1). In eukaryotes, AP sites are efficiently repaired by
excision repair processes (2-4). However, if not removed, they present
a block to the replication machinery. Thus, to maintain the continuity
of DNA during replication, AP sites encountered by the replication
machinery have to be bypassed. In the yeast Saccharomyces
cerevisiae, genes in the RAD6 epistasis group promote
replication through DNA lesions (5-7). The REV1, REV3, and
REV7 genes of this epistasis group are essential for damage-induced mutagenesis (8), including mutagenesis induced by AP
sites (3). The Rev1 protein has a deoxycytidyltransferase activity that
can incorporate a dCMP residue opposite an abasic site (9), and the
Rev3 and Rev7 proteins associate to form DNA polymerase
(10).
In vitro, the combination of Rev1 and Pol
promotes AP
bypass (9).
The yeast RAD30 gene, which belongs to the RAD6
epistasis group, encodes a DNA polymerase, Pol
, that has the unique
ability to efficiently replicate through a cis-syn
thymine-thymine (T-T) dimer; it does so correctly by inserting two
A residues across from the T-T dimer (11, 12). Human Pol
resembles
yeast Pol
in replicating through the T-T dimer with the same
efficiency and accuracy as through undamaged Ts (13). Consistent with
the error-free bypass of the T-T dimer, inactivation of yeast and human
Pol
causes UV hypermutability (7, 14, 15). Patients with the variant
form of xeroderma pigmentosum are defective in Pol
(16, 17), and as
a consequence, they suffer from a high incidence of UV-induced skin cancers.
In addition to the T-T dimer, yeast and human Pol
are able to bypass
the 8-oxoguanine (8-oxoG) lesion efficiently and accurately (18). In
contrast to eukaryotic polymerases
,
, and
, which preferentially incorporate an A opposite the 8-oxoG lesion, Pol
predominantly inserts a C opposite the 8-oxoG lesion (18). Also, yeast
and human Pol
are able to bypass the O6-methylguanine
(m6G) lesion, and they incorporate a C or a T residue opposite this
lesion (19).
For DNA polymerases lacking the proofreading 3'
5' exonuclease
activity, the fidelity for nucleotide insertion depends upon the
requirement of the polymerase active site for correct Watson-Crick base
pairing geometry and upon the ability of bases to form proper hydrogen
(H) bonding. Most DNA polymerases are highly sensitive to geometric
distortions in DNA (20), and their fidelity is affected more severely
by the disruption of optimal geometry than by H bonding between
base pairs (21, 22). As a consequence, they are unable to incorporate
nucleotides opposite lesions that distort the DNA helix.
Previously, we suggested that the ability of Pol
to bypass lesions,
such as the T-T dimer, 8-oxoG, and m6G, results from an unusual
tolerance of its active site for the distorted template geometries of
these lesions. To further assess the range of template lesions
tolerated by Pol
, here we examine the bypass of an abasic site, a
prototypical noninstructional lesion. We find that Pol
inserts
nucleotides opposite the AP site very poorly, and it also extends from
the inserted nucleotide very inefficiently. These results suggest that
Pol
requires the presence of template bases opposite both the
incoming nucleotide and the primer terminus to catalyze efficient
nucleotide insertion.
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MATERIALS AND METHODS |
DNA Polymerase Reactions--
Standard DNA polymerase reactions
(10 µl) contained 40 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 1 mM dithiothreitol, 100 µg/ml bovine serum albumin, 10% glycerol, 20 nM 5'
32P-labeled oligonucleotide primer annealed to an
oligonucleotide template, and dNTP in the concentrations indicated in
the figure legends. Reactions were initiated by adding yeast or human
Pol
at the concentrations indicated in the figure legends. After
incubation for 5 min at 30 °C, reactions were terminated by the
addition of 40 µl of loading buffer containing 20 mM
EDTA, 95% formamide, 0.3% bromphenol blue, and 0.3% cyanol blue. The
reaction products were resolved on 10 or 20% polyacrylamide gels
containing 8 M urea and were dried before autoradiography
at
70 °C with intensifying screens. A Molecular Dynamics STORM
phosphorImager and ImageQuant software were used for quantitation. DNA
substrates S-1 and S-2 were generated by annealing the 75-nt oligomer
template (N75AP, 5'-AGCTACCATGCCTGCCTCAAGAGTTCGTAA0ATGCCTACACTGGAGTACCGGAGCATCGTCGTGACTGGGAAAAC-3'), which contained an AP site (a tetrahydrofuran moiety; Midland Co.) at
the underlined 0 at position 31 or a nondamaged G residue at
this position, respectively, to the 32-nt 5' 32P-labeled
oligomer primer (N4456, 5'-GTTTTCCCAGTCACGACGATGCTCCGGTACTC-3'). For steady-state kinetic analysis, DNA substrates S-3, S-4(G), S-4(A),
S-4(T), and S-4(C) were generated by annealing a 52- nt oligomer
template
(5'-TTCGTATAATGCCTACACT0GAGTACCGGA GCATCGTCGTGACTGGGAAAAC-3'), which contained an AP residue at the underlined position 20 to the
32-nt and four different 33-nt 5' 32P-labeled oligomer
primers (N4456 or oligonucleotides that contain N4456 with one
additional G, A, T or C residue at its 3'-end, respectively). DNA
substrates S-5(G), S-5(A), S-5(T), and S-5(C) were generated by
annealing the N75AP oligomer template to four different 45-nt 5'
32P-labeled oligomer primers that contain oligomer N4309
(5'-GTTTTCCCAGTCACGACGATGCTCCGGTACTCCAGTGTAGGCAT-3') with one
additional G, A, T, or C residue at its 3'-end. In nondamaged control
DNA substrates the complementary bases were used instead of the AP
site. The sequence of the DNA substrate containing the 18-nt template
oligomer annealed to the 12-nt primer is shown in the figures.
Steady-state Kinetic Analyses--
Steady-state kinetic analysis
for each deoxynucleotide incorporation opposite the AP site was done as
described previously (23-25). Analyses of primer extension from this
lesion were carried out in a similar manner, except that only the
correct incoming deoxynucleotide was added to the reaction and the
primer varied at the 3' primer end. Briefly, Pol
was incubated with
increasing concentrations of a single deoxynucleotide (0-1000
µM) for 1 min under standard reaction conditions. Gel
band intensities of the substrates and products were quantitated by
PhosphorImager. The percentage of primer extended was plotted as a
function of dNTP concentration, and the data were fit by nonlinear
regression using SigmaPlot 5.0 to the Michaelis-Menten equation
describing a hyperbola, v = (Vmax × [dNTP]/(Km + [dNTP]). Apparent Km and
Vmax steady-state parameters were obtained from
the best fit.
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RESULTS |
An Abasic Site Is a Block to Yeast Pol
--
To determine
whether yeast Pol
replicates past an abasic site in template DNA, we
used a running start DNA substrate containing a single AP site in a
75-nt template DNA in which the DNA polymerase must synthesize 12 nt
before encountering the lesion. DNA synthesis reactions were carried
out in the presence of a 4-fold excess of DNA substrate over Pol
and
from low to higher dNTP concentrations (0.5-50 µM).
yPol
replicated through the AP site very poorly, and even at 50 µM dNTP, only ~5% translesion synthesis
occurred (Fig. 1A, lanes
5-8) compared with synthesis on a template containing a normal G
residue (Fig. 1A, lanes 1-4). Furthermore,
yPol
exhibits two strong stall sites, one right before the lesion
and the other opposite the lesion, indicating an inhibition of
insertion across from the AP site as well as an inhibition of extension
from the nucleotide inserted opposite the lesion. A stall site at the
position just after the AP site indicates that elongation opposite the 5' residue next to the AP site is also inhibited.

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Fig. 1.
Translesion DNA synthesis
activities of yeast Pol on templates
containing an AP site. A, running start DNA synthesis
past an AP site by yPol . A portion of the DNA substrate
adjacent to the primer-template junction is shown for the 75-nt
template and the 32-nt, 5' 32P-labeled primer. The
position of the undamaged G residue (in the S-2 substrate)
(lanes 1-4) or the AP site (in the S-1 substrate)
(lanes 5-8) in the template is indicated by
asterisks. yPol (5 nM) was incubated with the
DNA substrate (20 nM) in the presence of increasing
concentration (0.5-50 µM) of each of four dNTPs. The
amount of synthesis past the undamaged G or AP site is indicated.
B, identification of nucleotides incorporated opposite the
AP site by yPol . Standing start reactions were carried out on 18-nt
templates containing either a G (lane 5) or an AP site
(lanes 6-10) at position 13. The asterisks
indicate the position of the G or the AP site in the template.
Reactions were carried out in the presence of all four dNTPs (100 µM each, lanes 5 and 6), or in the
presence of a single dNTP (100 µM, lanes
7-10), 20 nM DNA substrate, and 40 nM
yPol . Reaction mixtures were resolved on a 20% denaturing
polyacrylamide gel; electrophoretic mobilities of the 18- and 13-nt
synthetic oligomers representing full-length products and primer
extended by one nucleotide, respectively, and containing either a C, A,
T, or G at position 13 are shown in lanes 1-4,
respectively. N, all four dNTPs.
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Deoxynucleotides Inserted Opposite the AP Site by Yeast
Pol
--
To identify the deoxynucleotide inserted opposite the AP
site, we assayed yPol
on an 18-nt template having either a G or an
AP site at position 13 from the 3' end in the template, primed with a
12-nt primer (Fig. 1B) in the presence of a single or all four nucleotides. As markers, we used the 13- and 18-nt oligomers representing a primer extended by one nucleotide and full-length products, respectively, and containing a C, A, T, or G residue at
position 13, which can be distinguished by their relative
electrophoretic mobility on 20% polyacrylamide gels (Fig.
1B, lanes 1-4). To facilitate bypass, we used
high yPol
as well as high dNTP concentrations, which on the
undamaged G template resulted in 100% synthesis to the end of the
template DNA (Fig. 1B, lane 5). Even under these forcing conditions, yPol
carried out almost no AP bypass, and only
nucleotide incorporation opposite the AP site without further extension
was observed (Fig. 1B, lane 6). In the presence
of all four dNTPs, yPol
inserted primarily a G residue (95%) across from the AP site (Fig. 1B, lane 6). With only a
single nucleotide present besides a G residue, yPol
also
incorporated an A, and T was inserted very weakly opposite the AP site
(Fig. 1B, lanes 7-10).
Steady-state Kinetic Analyses of Nucleotide Insertion Opposite the
AP Site and of Subsequent Extension by Yeast Pol
--
Next we
measured the kinetics of nucleotide insertion and extension during DNA
synthesis past the AP site. To determine the frequency of nucleotide
incorporation by yPol
, we measured the Km and
Vmax steady-state kinetic parameters (23-25)
for all four incoming dNTPs opposite a template AP site. For purposes of comparison, the kinetic parameters opposite nondamaged template residues were measured as well. yPol
was incubated with the DNA substrate and with increasing concentrations of one of the four deoxynucleotides. The pattern of deoxynucleotide incorporation by
yPol
opposite an AP site is shown in Fig.
2A. The Km and Vmax parameters were determined and used to
calculate the percentage of each nucleotide incorporated opposite the
AP site (Table I). yPol
incorporated
59% G, 31% A, 7% T, and 3% C opposite the AP site. This
analysis indicates that yPol
incorporates a G opposite the AP site
with a 2-fold higher efficiency than A. Importantly, however, yPol
inserts a G opposite the AP site about 1,000-fold less efficiently than
the insertion of G opposite C (Table I). The other nucleotides were
inserted even less efficiently (Table I). The substantially lower
efficiency of nucleotide incorporation opposite an AP site relative to
a nondamaged template residue results from a 1,000-10,000-fold
increase in the Km for dNTP (Table I).

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Fig. 2.
Insertion and extension reactions catalyzed
by yeast Pol on an AP site containing DNA
template. A, deoxynucleotide incorporation across from a
template AP site. yPol (5 nM) was incubated with the S-3
primer-template DNA substrate (20 nM) and increasing
concentrations (0-1000 µM) of a single deoxynucleotide
(dGTP, dATP, dTTP, or dCTP) in standard reaction buffer. The quenched
samples were analyzed by 10% denaturing polyacrylamide gel
electrophoresis. B, extension of primers containing a G, A,
T, or C residue opposite the AP site in the template by yPol .
Primers differing only in the last nucleotide at the 3'-end were
annealed separately to an AP site containing DNA template as shown on
the top. Reactions were performed in the presence of
increasing dATP concentrations (0-1000 µM) as described
for panel A.
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For lesion bypass to occur, it is important that, after incorporating a
nucleotide opposite the lesion, a polymerase extend the primer beyond
the lesion. To examine the efficiency of extension past the AP site,
the steady-state kinetic parameters of the addition of the next correct
deoxynucleotide by yPol
on substrates in which the 3' terminus of
the primer is paired with an AP site were measured. Fig. 2B
presents the pattern of extension from G, A, T, or C when paired with
an AP site. From the pattern of extension from these different 3'
termini, the Km and Vmax
parameters were obtained and used to calculate the efficiency (Vmax/Km) of extension. The
ratio of extension opposite from the AP site was: G:A:T:C = 49:37:9:5 (Table II), which indicates that yPol
extends from a G or an A opposite the AP site about equally well. yPol
, however, extends from G or A quite
inefficiently, as the efficiency
(Vmax/Km) of extension in
both cases was reduced by almost 1000-fold that from the opposite
nondamaged complementary bases (Table II). The lower efficiency of
extension from bases opposite an AP site relative to the extension from bases opposite a nondamaged residue was also because of a
500-2000-fold increase in the Km for dNTP (Table
II). Hence, yPol
is very inefficient in inserting nucleotides across
from an AP site as well as in extending from the nucleotide
inserted.
Inefficient Nucleotide Insertion Opposite an AP Site and
Inefficient Extension of Inserted Nucleotide by Human Pol
--
We
also examined the ability of the human DNA polymerase
(hPol
) to
bypass the AP site. Like yPol
, hPol
bypasses the AP site very
inefficiently. hPol
also exhibits two stall sites, one right before
the AP site and the other opposite the lesion, indicating that there is
inhibition of deoxynucleotide insertion opposite the AP site as well as
inhibition of extension from this lesion (data not shown). The
extremely restricted ability of hPol
to bypass an AP site is further
reflected in its steady-state Km and
Vmax kinetic values. The kinetics of insertion of a single deoxynucleotide opposite an AP site and the kinetics of
addition of the next correct nucleotide to various 3'-primer termini
situated across from the AP site were determined as a function of
deoxynucleotide concentration. hPol
also inserts a G somewhat better
than an A opposite the AP site. However, hPol
inserts these
nucleotides opposite the AP site ~103-fold less
efficiently than opposite the nondamaged complementary base, because of
a 600-2,500-fold increase in the Km for dNTP (Table
III). The order and the ratio of
deoxynucleotide insertion opposite the AP site by hPol
were
G:A:C:T ~ 13:10:1.5:1 (Table III). hPol
also extends
inefficiently from the nucleotide inserted opposite the AP site, and
the order and the frequency of extension from different 3'-terminal
deoxynucleotides paired with the AP site were A:G:C:T ~ 5:2:2:1
(Table IV). Thus, human Pol
inserts G
slightly better than A opposite the AP site, but it is 2.5-fold more
efficient at extending from an A opposite the AP site than from a G
opposite this lesion. The poor extension efficiency of hPol
is again
because of a 500-1500-fold increase in the Km for
dNTP (Table IV).
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DISCUSSION |
Pol
is unique among eukaryotic DNA polymerases in its ability
to bypass a cis-syn T-T dimer and an 8-oxoG lesion
efficiently and accurately. Although a cis-syn T-T dimer
disrupts the DNA helix, this distortion does not affect the ability of
two Ts in the dimer to base pair with As (26-28). 8-oxoG in the
syn conformation mimics T and has the correct geometry to
base pair with A, whereas 8-oxoG in the anti-conformation
base pairs with C (29-32). The template strand, however, is
significantly distorted in the vicinity of the lesion in the 8-oxoG·C
base pair (29-32). Both yeast and human Pol
incorporate As opposite
the two Ts of the T-T dimer with the same efficiency and accuracy as
opposite undamaged Ts (12, 13). In contrast with eukaryotic replicative
polymerases
,
, and
, which all bypass 8-oxoG by incorporating
an A opposite the lesion, Pol
bypasses 8-oxoG by inserting
predominantly a C opposite the lesion (18). These and other
observations (19) have suggested that Pol
is refractory to geometric
distortions conferred upon DNA by these lesions.
Here we examine the ability of Pol
to bypass an AP site. An abasic
site is a prototypical noninstructional DNA lesion. NMR studies have
indicated that DNA containing an A opposite the AP site retains all
aspects of B-form DNA, and the unpaired A and the abasic residue lie
inside the helix (33-35). The A is held well in the helix as if paired
with T, and the melting temperature of the A· AP site is the same as
that of the A·T base pair (33-35). At low temperatures, a G opposite
the AP site is also predominantly intrahelical (35). However, when a
pyrimidine is positioned opposite the AP site, both the pyrimidine and
the abasic sugar are extrahelical, and the helix collapses (35). Many
DNA polymerases insert an A opposite the AP site (36), presumably
because the geometry of an A opposite an AP site closely resembles an
A·T base pair.
As revealed from steady-state kinetic analyses of nucleotide insertion
and extension, both yeast and human Pol
incorporate nucleotides
opposite the AP site very inefficiently, and they are also highly
inefficient in subsequent extension of the primer. This suggests that
Pol
requires the presence of template bases opposite both the
incoming nucleotide and the primer terminus to catalyze efficient
nucleotide incorporation. In the absence of either of these template
bases, either the enzyme or the enzyme-bound DNA substrate may adopt a
conformation that is not conducive to nucleotide incorporation. Such a
conformational alteration could then result in weaker dNTP binding to
the enzyme-DNA complex resulting in the substantial increase in the
Km for dNTP observed in both the incorporation
opposite an AP site and the extension from bases opposite the AP site.
Although the results reported here provide no compelling evidence for a
role of Pol
in AP bypass, they do not exclude the possibility that
association with accessory factors modifies the damage bypass ability
of this polymerase. In Escherichia coli, RecA stimulates the
DNA synthesis efficiency of the UmuCD' complex (PolV) 15,000-fold, and
the increased efficiency is reflected mainly in the
Km reduction for dNTPs (37). By reducing the
Km for dNTPs, accessory factors may facilitate AP bypass by promoting nucleotide incorporation opposite the lesion by
Pol
.