Repair of apurinic/apyrimidinic (AP) sites by
mammalian cell extracts was compared using circular and linear DNA
substrates. Extracts prepared from DNA polymerase
(pol
)-proficient mouse fibroblasts repaired AP sites on both
circular and linear DNA. However, extracts from the isogenic
pol
-knockout cells repaired AP sites on circular DNA but not
efficiently on linear DNA. The circularity-dependent repair
by the pol
-knockout cell extract was completely inhibited by
anti-proliferating cell nuclear antigen (PCNA) antibody but fully
restored by addition of purified PCNA. Pretreatment of the linear DNA
with AP endonuclease did not improve repair, indicating that impairment
of AP site repair on linear DNA by pol
-knockout cell extracts is not
due to inefficiency of damage incision but rather to deficiency at the
subsequent steps. These results indicate that AP sites can be repaired
on circular DNA by the PCNA-dependent pathway in addition
to the pol
-dependent pathway and that the
PCNA-dependent repair mechanism is poorly functional on
linear DNA in vitro.
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INTRODUCTION |
Base excision repair is a major mechanism by which cells correct
bases with small modifications,
AP1 sites, and single-strand
breaks (1). These types of DNA damage are spontaneously generated
through normal cellular metabolism and also are caused by exogenous
agents such as ionizing radiation and alkylating and oxidizing agents.
Thus, they belong to a group of the most abundant lesions in living
cells. A typical process for base excision repair consists of five
sequential reactions (2): (i) the modified base is removed by a
specific DNA-N-glycosylase; (ii) the resultant AP site is
incised at its 5
side by an AP endonuclease; (iii) a deoxyribose
phosphate (dRP) is excised from the 5
-incised site; (iv) the gap is
filled by DNA synthesis; and (v) the DNA strand is sealed by ligation.
Several protein factors responsible for some steps in this process have
been identified, including a group of DNA-N-glycosylases and
AP endonucleases. Regarding the DNA synthesis step, however, it has not
yet been established which enzymes are responsible for this step.
Higher eukaryotes have five classes of DNA polymerases in nuclei,
,
,
,
, and
(3). Among them, pol
has long been considered to be involved in base excision repair. Two recent observations strongly support this implication. First, a mouse fibroblast cell line
in which both alleles of the pol
gene are disrupted exhibits hypersensitivity to alkylating agents (4). Second, pol
has beside
its DNA polymerase activity, another enzymatic activity for the
excision of dRP residues, an indispensable step for base excision
repair (5). On the other hand, two studies using in vitro
repair systems demonstrated that base excision repair can proceed by
another pathway that requires PCNA as an essential factor (6, 7).
PCNA plays an essential role in DNA replication (8, 9) and in
nucleotide excision repair (10, 11), and it has been shown recently to
be involved in mismatch repair (12). It is a protein of approximately
29 kDa that forms a homotrimer with a torus structure. Double-stranded
DNA can pass through the inside cavity of the PCNA trimer. Such loading
of PCNA onto DNA is facilitated by the replication factor C (RF-C) in
an ATP-dependent manner, resulting in a PCNA/RF-C complex
known as the PCNA clamp (for review, see Ref. 13). The formation of the
PCNA clamp is a prerequisite to efficient DNA synthesis by DNA
polymerase
(pol
) or polymerase
(pol
). Because PCNA
stimulates the activities of pol
and pol
but not polymerase
or polymerase
, the alternative pathway for base excision repair
should use pol
and/or pol
at its DNA synthesis step. In contrast,
several studies which also used in vitro repair systems
indicated that pol
was the exclusive DNA polymerase responsible for
base excision repair (4, 14, 15). We noticed that the studies
demonstrating the PCNA-dependent pathway used circular DNA
substrates, whereas linear DNA substrates were used in the studies
supporting the pol
-dependent pathway in exclusion of the
PCNA-dependent pathway. Podust et al. (16)
reported that PCNA could be loaded more stably on circular DNA than on
linear DNA in vitro. Unstable loading of the PCNA onto
linear DNA is due to the falling off of the PCNA clamp from linear DNA
ends after sliding along it. If this property of PCNA-DNA binding
applies to base excision repair, linear DNA substrates may not support the PCNA-dependent pathway. To test this possibility, we
compared the repair efficiencies on circular and linear DNA side by
side using extracts prepared from wild-type and pol
-knockout mouse cells. We show here that DNA substrates that do not have free ends are
preferable to detect PCNA-mediated in vitro repair.
 |
EXPERIMENTAL PROCEDURES |
Cell Lines--
The wild-type mouse embryonic fibroblast cell
line and the matched littermate pol
-knockout cell line (4) were
maintained in Glutamax I medium (Life Technologies, Inc.) supplemented
with 10% fetal bovine serum (Atlanta Biologicals), 100 units/ml
penicillin, 100 µg/ml streptomycin, and 80 µg/ml hygromycin B
(Sigma). The cells were cultured in monolayer in a humidified incubator
with 10% CO2 at 34 °C. CHO-K1 and its double-strand
break repair-deficient derivative, xrs-5, were maintained in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% fetal bovine
serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine in a humidified incubator with
5% CO2 at 37 °C.
Preparation of Cell Extracts--
Cells were cultured in 175 cm2 flasks and incubated overnight to reach mid-exponential
growth phase. The cells were then washed three times with ice-cold
phosphate-buffered saline and resuspended at 106 cells/20
µl in Buffer I (10 mM Tris-Cl, pH 7.8, and 200 mM KCl). After the addition of an equal volume of Buffer II
(10 mM Tris-Cl, pH 7.8, 200 mM KCl, 2 mM EDTA, 40% glycerol, 0.2% Nonidet P-40, 2 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl
fluoride, 10 µg/ml aprotinin, 5 µg/ml leupeptin, 1 µg/ml
pepstatin), the cell suspension was rocked at 4 °C for 1 h and
then centrifuged at 16,000 × g for 10 min. The
supernatant was recovered and stored in small aliquots at
80 °C.
Preparation of DNA Substrates Containing an AP
Site--
Covalently closed circular DNA (cccDNA) which carried
either a uridine or a synthetic AP site analog
(3-hydroxy-2-hydroxymethyltetrahydrofuran) was prepared as described
previously (6). In prelabeled cccDNA, 32P was
incorporated at the position several nucleotides away from the lesion.
Linear DNA was prepared by digestion of the cccDNA with
PvuII, which generated a 382-base pair fragment
carrying the AP site in its middle position and a 2.8-kilobase fragment with no AP site. For natural AP site repair assays, the DNA containing a uridine residue was treated with uracil-DNA glycosylase
(Perkin-Elmer) immediately before the repair reaction as described
previously (6) to create a natural AP site.
DNA Repair Assay--
A standard repair reaction was carried out
at 25 °C with 10 ng of a DNA substrate (200-1000 cpm, 5 fmol AP
sites) and 10 µg of the cell extract protein in a 20-µl reaction
mixture (20 mM HEPES-KOH, pH 7.5, 10 mM
MgCl2, 135 mM KCl, 1 mM
dithiothreitol, 2 mM ATP, 20 µM each of four
dNTPs, 2 mM NAD, 40 mM phosphocreatine, and 1 unit of creatine phosphokinase). When indicated, repair factors were
added to the cell extracts immediately before the reaction was started.
In experiments for neutralizing PCNA, the antibody AK specific for PCNA
(37.5 µg/ml) (generously provided by Dr. M. Miura, Tokyo Medical and
Dental University) was incubated with the cell extracts on ice for 20 min before the repair reaction. At indicated times the reaction was
stopped by the addition of sodium dodecyl sulfate to a final
concentration of 0.4% and 2 µg of proteinase K followed by a 30-min
incubation at 37 °C. The DNA was recovered by phenol:chloroform
extraction and ethanol precipitation and digested with AP endonuclease
and PvuII (for cccDNA only). The DNA was then subjected
to electrophoresis on an 8 M urea-containing 6%
polyacrylamide gel. Repaired and unrepaired DNA fragments were detected
by autoradiography, and the amounts of radioactivities were quantified
with a Fuji BAS 1000 phosphorimager.
Detection of Uncut AP Sites--
After the repair reaction, DNA
was digested with KpnI and PvuII (for cccDNA
only) for 1 h at 37 °C and subjected to electrophoresis on an 8 M urea-containing 6% polyacrylamide gel. KpnI
can cleave only the repaired DNA substrate but not the unrepaired DNA
substrate, which has an AP site in the KpnI restriction
site. The 382-nucleotide fragment that carried an intact AP site and
the 131-nucleotide and 129-nucleotide fragments which were generated by
the AP endonuclease digestion during the repair reaction or by the
KpnI digestion of the repaired product were detected by
autoradiography, and the amounts of radioactivities were quantified
with a Fuji BAS 1000 phosphorimager.
 |
RESULTS |
Procedures for Cell Extract Preparation--
Most of the studies
dealing with in vitro DNA repair assays with mammalian
cultured cells utilize extracts prepared by the procedures described by
Manley et al. (17) with some modifications. This technique
is time consuming and requires large amounts of cells. We prepared
extracts from detergent-lysed cells by the procedures described by
Tanaka et al. (18) with a few modifications (see under
"Experimental Procedures" for details). These procedures allowed us
to obtain from a million cells an amount of cell extract sufficient for
30 repair reactions. In a preliminary experiment, we compared the
repair activity of cell extracts prepared by this method with that of
the extracts prepared as described by Manley et al. (17).
These extracts were made from mouse pol
-knockout cells and their
isogenic wild-type cells. Repair assays were conducted on AP
site-containing circular DNA. AP sites are common intermediate products
generated during base excision repair and are efficiently repaired by
Xenopus laevis ovarian extracts (6) and mammalian cell
extracts (7). It has been demonstrated with a reconstituted repair
system using X. laevis proteins that a synthetic AP site analog, 3-hydroxy-2-hydroxymethyltetrahydrofuran, is mostly repaired by
the PCNA-dependent pathway, whereas the natural AP site is repaired by both the pol
-dependent pathway and the
PCNA-dependent pathway (6). Therefore, we also compared
repair of these two types of lesions. As shown in Table
I, both cell extracts prepared from
wild-type and pol
-knockout cells were able to repair AP sites,
although less efficiently by the latter. This result indicates that AP
sites were repaired, at least in part, by a mechanism independent of
pol
in these mouse cell extracts. In each case, the extract prepared
by the method of Tanaka et al. provided higher repair
activity than the extract prepared by the method of Manley et
al. Therefore, the subsequent experiments in this study were carried out using cell extracts prepared by the procedure based on the
detergent cell lysis.
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Table I
Comparison of the repair activity of cell extracts prepared by two
different methods
Extracts were prepared from indicated cells by the method of Tanaka
et al. (18) (I) or the method of Manley et al.
(17) (II). Repair reactions with circular DNA carrying the indicated lesions were carried out for 30 min at 25 °C as described under "Experimental Procedures."
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Influence of DNA Structure on AP Site Repair--
To evaluate more
accurately the repair activity of each extract, a time course of the
repair was measured. On circular DNA, the synthetic AP site analog was
repaired at the same rate by both wild-type and pol
-knockout cell
extracts (Fig. 1A). In
contrast, the natural AP site was more efficiently repaired by
wild-type cell extracts (Fig. 1B), indicating that a portion
of repair in the wild-type cell extract was mediated by a
pol
-dependent pathway. The initial rate of natural AP
site repair by the pol
-knockout cell extract was similar to that
observed for the synthetic AP site repair by both extracts. Thus, the
difference of the natural AP site repair between the two extracts could
be due to preferential repair of the natural AP sites over the
synthetic AP sites by the pol
-dependent mechanism. As
previously reported, pol
can excise a dRP residue from the
5
-incised AP site by its own dRP lyase activity (5). However, pol
cannot excise a dRP residue from the synthetic AP site analog, because
this lesion is resistant to
-elimination. It was observed that
synthetic AP sites were not efficiently repaired by the
pol
-dependent pathway (6). On linear DNA, however, only
the wild-type cell extracts were able to repair efficiently the natural
AP site and to a lesser extent the synthetic AP site analog (Fig. 1,
C and D). The complete lack of DNA repair with
the pol
-knockout cell extracts on linear DNA indicates that only the
pol
-dependent pathway is functional on this DNA. Thus,
the alternative pathway cannot repair either type of AP sites on linear
DNA. The effect of DNA linearization on AP site repair appeared to have
an inverse correlation with the length of the linear DNA. Although the
PvuII-digested DNA (382 base pairs) was not repaired at all
by pol
-knockout cell extracts (Fig. 1), the
XmnI-linearized DNA (3.2 kilobases) was repaired by the same
extracts with approximately 50% efficiency of that of circular DNA
(data not shown).

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Fig. 1.
Influence of DNA structure on AP site
repair. Repair reactions were carried out by incubating circular
or linear DNA carrying either a natural AP site or a synthetic AP site
analog with the extract from wild-type mouse fibroblasts
(wt) or pol -knockout (pol / ) cells for
0, 15, 30, 60, and 90 min as described under "Experimental
Procedures." A, repair of the synthetic AP site on
circular DNA. B, repair of the natural AP site on circular DNA. C, repair of the synthetic AP site analog on linear
DNA. D, repair of the natural AP site on linear DNA.
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According to the previously reported studies (6, 7), this alternative
pathway for base excision repair should proceed through the
PCNA-dependent DNA synthesis. To test whether this is the
case in the present repair system with mouse cell extracts, we used an
anti-PCNA antibody (AK) known to have a neutralizing activity for the
PCNA-dependent DNA synthesis (19). When incubated for 20 min with the cell extracts prior to the reaction, this antibody
completely inhibited the repair reaction carried out by pol
-knockout
cell extracts on circular DNA (Fig. 2).
Moreover, this repair was fully restored by addition of 500 ng of PCNA
to the reaction. These results clearly indicated that PCNA is required for the alternative pathway in the pol
-knockout cell extracts. The
repair of the synthetic AP site analog by the wild-type cell extracts
was only partially inhibited. This is because this synthetic lesion can
be repaired by the pol
pathway, although in a less efficient manner.
The synthetic AP site analog cannot be excised by pol
, because this
lesion is refractory to
-elimination (5). However, it is reported
that FEN1 (also known as DNaseIV) can excise modified AP sites which
cannot be removed by
-elimination (20). Therefore, the excision step
of the synthetic AP sites was probably catalyzed by a FEN1-like enzyme
present in the cell extracts. Once this step is accomplished, pol
should ensure the completion of the repair. We indeed observed that
addition of FEN1 to the reconstituted system for the
pol
-dependent AP site repair rescued the repair of
synthetic AP sites.2 A
similar inefficient repair of the synthetic AP site analog by the
pol
-dependent pathway is also shown in Fig.
1C (compare with Fig. 1D). The AK antibody did
not inhibit at all the natural AP site repair by the wild-type cell
extracts, because this repair can be catalyzed by the
pol
-dependent pathway without restriction. Taken
together, the PCNA-dependent AP site repair can proceed in vitro on circular DNA but not efficiently on linear
DNA.

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Fig. 2.
Inhibition of repair by anti-PCNA
antibody. Wild-type cell extracts and pol / cell extracts
were preincubated with AK antibody when indicated and used for repair
reactions of circular DNA carrying either a synthetic AP site analog
(A) or a natural AP site (B) as described under
"Experimental Procedures." When indicated, 500 ng of PCNA were
added immediately before the repair reaction. The repair reactions were
carried out for 1 h.
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Effect of Additional Repair Factors on AP Site Repair--
To
confirm further the differential effect of DNA structure between the
pol
-dependent pathway and the PCNA-dependent
pathway, we stimulated either one of the two pathways by adding
purified pol
or PCNA to the repair reactions with the cell extracts.
When we added 50 ng of rat pol
, the AP sites were mostly repaired in
any combination of the cell extracts and the DNA substrates (Fig.
3), indicating that the
pol
-dependent pathway was fully functional on both
circular and linear DNA substrates. On the other hand, the addition of
50 ng of mouse PCNA increased the repair efficiency on the circular DNA
but did not rescue the repair on the linear DNA (Fig.
4). Thus, the stimulating effect of
additional PCNA on AP site repair was limited to circular substrates.
The increase in AP site repair by additional PCNA was relatively small compared with that by additional pol
. This is probably because PCNA
may not be the only limiting factor for this repair pathway. Pol
,
pol
, and RF-C may also be limiting, as such large factors may not be
efficiently extracted from the nuclei during our cell extract
preparation procedures.

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Fig. 3.
Effect of additional pol on DNA
repair. Repair reactions were carried out for 1 h without
(open bars) or with 50 ng of rat pol (hatched
bars) in addition to the indicated extract as described under
"Experimental Procedures."
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Fig. 4.
Effect of additional PCNA on AP site
repair. Repair reactions were carried out for 30 min without
(open bars) or with 50 ng mouse PCNA (hatched
bars) as described under "Experimental Procedures" in addition
to the indicated extract.
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Inhibition of Base Excision Repair in Linear DNA Is Not Due to Ku86
Protein--
Recently, Calsou et al. (21) reported that
nucleotide excision repair was inhibited by double-strand breaks in a
cis-acting manner, and that this inhibition could be due to
the blocking by Ku proteins of the damage-specific incision. Ku
proteins which can bind as a heterodimer of Ku70 and Ku86 to
double-strand breaks are required for double-strand break DNA repair
and V(D)J recombination (22, 23). Calsou et al. (21)
observed that linearization of DNA substrates reduced the nucleotide
excision repair activity to 50% in the extracts from Ku-proficient
CHO-K1 cells but did not affect the repair in the extracts from
Ku86-deficient xrs-6 cells. We investigated a possible involvement of
Ku proteins in the AP site repair process on linear DNA. Cell extracts
were prepared from Ku86-deficient xrs-5 cells and the parental
Ku-proficient CHO-K1 cells, and their repair activities were compared
on linear and circular DNA carrying either a synthetic or a natural AP
site. Because the CHO cell lines were proficient in pol
activity,
natural AP sites on linear DNA were repaired in the extracts from these cells (Fig. 5). The repair of the
synthetic AP site analog was, in contrast, relatively inefficient on
linear DNA. This repair impairment was observed at similar levels in
both CHO-K1 and xrs-5 cell extracts. Furthermore, the AP site on linear
DNA was incised at the same rate by both cell extracts. The repair
reactions with CHO-K1 and xrs-5 cell extracts left 6 and 12% of AP
sites on linear DNA uncut (Table II),
indicating that 94 and 88%, respectively, of the DNA substrate was
incised by AP endonucleases contained in these cell extracts. On
circular DNA, the incision rate was higher than 95% for both cell
extracts. This result shows that the Ku86 protein did not inhibit the
incision step on either circular or linear DNA. We also examined AP
site repair of DNA pretreated with AP endonuclease. As shown in Fig.
6, preincision of AP sites did not
improve the repair of linear DNA by the pol
-knockout cell extracts,
indicating that the impairment of PCNA-dependent pathway on
linear DNA is not due to inefficient incision of AP sites. These
results ruled out the possible role of the Ku86 protein in the
inhibition of base excision repair on linear DNA in our system.

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Fig. 5.
Effect of Ku86 protein on DNA repair.
A, repair with CHO-K1 cell extracts. B, repair
with xrs-5 cell extracts. Repair reactions on circular DNA (open
symbols) or linear DNA (closed symbols) carrying either
a natural AP site (circles) or a synthetic AP site analog
(squares) were carried out for the indicated times as
described under "Experimental Procedures."
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Fig. 6.
Effect of AP endonuclease pretreatment on DNA
repair. Repair reactions were carried out for 60 min with
pol / cell extracts on the untreated DNA (open bars)
or on the DNA treated with AP endonuclease (hatched bars) as
described under "Experimental Procedures."
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 |
DISCUSSION |
In this report, we have demonstrated that AP sites can be repaired
by the pol
-dependent pathway and an alternative
pol
-independent pathway in extracts prepared from mouse
fibroblast-derived cells. The alternative pathway uses PCNA as one of
its essential factors. This PCNA-dependent pathway was
functional on circular DNA but not on linear DNA in vitro,
whereas the pol
-dependent repair reaction proceeded
efficiently on both circular and linear substrates. These results
resolve the controversy raised by the studies previously reported (4,
6, 7, 14, 15). In addition, it is clear that circular DNA should be
used as a substrate for in vitro repair assays when PCNA may
be involved in the reaction. Recently, Klungland and Lindahl (20)
reported that PCNA enhanced the pol
-dependent repair of
modified AP sites by stimulating the FEN-1 activity. They also observed
that pol
-neutralizing antibodies decreased this AP site repair to
5%, claiming pol
as the major enzyme for DNA synthesis in base
excision repair. Because they used a double-stranded oligonucleotide as
a substrate, it is likely that their results may underestimate the
contribution of PCNA-dependent DNA polymerases pol
/pol
to base excision repair. The study performed with
Xenopus purified enzymes demonstrated that pol
repaired
efficiently AP sites on circular DNA (6). The data reported here with
pol
-knockout cell extracts also suggest that DNA synthesis for the
PCNA-dependent repair should be attributed to pol
or
pol
.
One explanation for the mechanism of in vitro repair
inhibition on linear DNA was provided by Calsou et al. (21)
for nucleotide excision repair in which the damage-specific incision
may be blocked by Ku proteins through their binding to the DNA ends.
However, our results with xrs-5 cell extracts and those obtained with
the DNA preincised with AP endonuclease indicate that this was not the
case for the impairment of AP site repair that we observed on linear
DNA. Although the PCNA-dependent base excision repair and
nucleotide excision repair use a PCNA-dependent DNA
polymerase, either
or
, their incision mechanisms of damaged
sites are distinct from each other. Nucleotide excision repair requires at least 16 polypeptides for incision of damaged sites (24). This
incision step seems to be rate-limiting in the repair reaction with
mammalian cell extracts rather than the DNA synthesis step (25). In
contrast, AP endonuclease is sufficient by itself for incision of AP
sites in base excision repair.
Inability to repair AP sites on linear DNA by the
PCNA-dependent pathway results most likely from the
property of the PCNA clamp on DNA. Podust et al. (16)
demonstrated that the RF-C·PCNA complex assembled on gapped circular
DNA was easily lost once the DNA was linearized. Consequently, pol
with PCNA and RF-C cannot carry out DNA synthesis on linear DNA as
efficiently as on circular gapped DNA. Studies with X. laevis reconstituted systems suggest that RF-C is required in the
PCNA-dependent AP site repair (6). The efficient
utilization of circular DNA as a template for
PCNA-dependent reactions seems to be due to its structural character, for that circular DNA does not have free ends. In fact, Yao
et al. (26) reported that blocking of linear DNA ends with a
specific DNA-binding protein reduces the dissociation of the PCNA clamp
from DNA. This situation resembles the DNA repair reaction in living
cells. There is a model for in vivo repair in which DNA
repair may be associated with rearrangements in chromatin structure
including dissociation of nucleosomes around damaged sites (27).
Therefore, a free slide of the PCNA clamp could be limited within the
unfolded region of the damaged chromosomal DNA.
We thank M. Miura for the AK antibody and
S. W. Johnson, T. C. Hamilton, and A. T. Yeung for critical reading
of the manuscript.