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INTRODUCTION |
Base excision repair of uracil in DNA is initiated by uracil-DNA
glycosylase, which removes uracil by cleavage of the base-sugar glycosidic bond (1). The AP
site1 resulting from this DNA
glycosylase activity is then processed by AP endonuclease, DNA
polymerase
(pol
), and DNA ligase I (2, 3), resulting in the
replacement of one nucleotide. Processing of the AP site can also be
accomplished by a different subpathway of BER, resulting in a longer
DNA repair patch of several nucleotides (4, 5). A satisfactory
understanding of the enzymes participating in this "long patch" BER
and the mechanisms involved has not been achieved. It was shown that
long patch BER reconstituted with partially purified components
depended on PCNA (4) and that long patch BER in cell extract is
sensitive to PCNA antibody (5). It was suggested that DNA polymerases
or
are involved in the gap-filling step during long patch BER (4, 5) because these enzymes are known to be stimulated by PCNA and
these polymerases are proficient in reconstituted long patch BER
systems (6, 7). Alternatively, if the role of PCNA in long patch BER is
limited to the stimulation of FEN1 cleavage of a flap DNA substrate
(8-10), other polymerases may also be considered as participants in
long patch BER. There are observations suggesting that pol
can
operate during long patch BER. Both DNA polymerases
and
can
function in long patch BER reconstituted with purified proteins (7),
and it was demonstrated that cell extract-mediated long patch BER is
inhibited by antibody to pol
(7). Also, pol
null cell extract
does not repair a reduced abasic site in a linear DNA substrate that is
repaired through long patch BER in pol
-containing wild-type cell
extract (11). Further, the absolute amount of long patch BER activity (substrate repaired per mg of extract protein) in the pol
null cell
extract is less than that in the isogenic wild-type cell extract.2 Taken together,
these results suggest that pol
, in addition to its role in
single-nucleotide patch repair, could play an important but yet unknown
role in long patch BER. In light of this background information, we
directly addressed the role of pol
in the excision process of long
patch BER performed by mammalian cell extracts.
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MATERIALS AND METHODS |
Cells and Extracts--
Normal human lymphoid cell line AG9387
was obtained from the Human Genetic Mutant Cell Repository (Coriell
Institute, Camden, NJ). Cells were grown in medium recommended by the
supplier. The DNA pol
-knockout mouse fibroblasts and the isogenic
wild type cell lines were grown as described (12). Whole cell extracts were prepared from 3-5 g of cells by the method of Manley et
al. (13) and dialyzed overnight against buffer containing 25 mM Hepes-KOH, pH 7.9, 2 mM dithiothreitol, 12 mM MgCl2, 0.1 mM EDTA, 17%
glycerol, and 0.1 M KCl. Extracts were aliquoted and stored at
80 °C.
Proteins and Antibodies--
Human DNA pol
was purified as
described (14). Polyclonal antibodies against human pol
were raised
in rabbit and were affinity-purified on a pol
-Sepharose column.
Construction of Closed Circular M13 DNA Containing a Single
Uracil Residue--
Double-stranded closed circular DNA containing
single uracil (U-DNA) was constructed as described (15) by priming
single-stranded M13 DNA with the 5'-labeled oligonucleotide
32pUCGGCCGATCAAGCTTATTGGGTACCG for internally
labeled uracil-containing substrate and
32pCCGGCCGATCAAGCTTATTGGGTACCG for the control
DNA substrate.
Excision Assay--
Standard 10-µl reactions contained 50-100
ng of internally labeled U-DNA, 45 mM Hepes-KOH (pH 7.8),
70 mM KCl, 7.5 mM MgCl2, 1 mM dithiothreitol, 0.4 mM EDTA, 2 mM ATP, 3.4% glycerol, 50 µM each of dGTP,
dATP, TTP, dCTP, 1 µg of 30-mer single-stranded oligonucleotide (as a
carrier), and 20 µg of a cell extract protein. DNA repair synthesis
reactions were carried out at 32 °C for the indicated times. After
the reaction, excision products were stabilized by the addition of 0.5 M NaBH4 to a final concentration of 0.1 M and incubated for 30 s on ice. Then 12 µl of
formamide-dye solution was added, and the reaction products were
separated by electrophoresis on a 20% denaturing polyacrylamide gel.
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RESULTS |
Excision Products Generated by Human Cell Extract--
To assess
the role of pol
in long patch BER of uracil-DNA (U-DNA), we
analyzed BER excision products formed in cell extracts. A closed
circular plasmid DNA substrate containing a 32P-labeled
phosphate group immediately 5' to a solitary uracil-guanine base pair
(Fig. 1a) was prepared. To
release the labeled phosphate from the substrate, enzymes in a cell
extract must first excise uracil and then incise the phosphodiester
bond 5' to the arising AP site. Next, the labeled phosphate is released
either as the 5'-deoxyribose phosphate residue (dRP) or as dRP moiety
attached to a short oligonucleotide produced after strand displacement and flap incision (dRP-oligo). Under our standard reaction conditions, >70% of the U-DNA substrate was consumed in 60 min (Fig. 1,
a and b). In addition to release of a dRP, which
migrated very quickly out of the gel, there was release of a
predominant dRP-oligo product that almost co-migrated with a
trinucleotide marker (Fig. 1b). A control DNA substrate with
a normal cytosine-guanine base pair at the same position as the
uracil-guanine lesion was not degraded (Fig. 1b, lane
C).

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Fig. 1.
Long patch excision products generated during
repair of U-DNA by human cell extract. a, U-DNA
substrate containing a single-uracil residue and labeled phosphate
group (in bold) 5' next to uracil is shown. b,
100 ng of internally labeled U-DNA was incubated with 20 µg of human
whole cell extract at 32 °C for the indicated time period. Control
reaction (lane C) containing 100 ng of internally labeled
substrate DNA with a regular C:G base pair at the same position was
incubated with 20 µg of human whole cell extract at 32 °C for
1 h. After the incubation, reaction products were reduced with
sodium borohydride and analyzed by electrophoresis on 20%
polyacrylamide gel. c, the 5'-deoxyribose phosphate
oligonucleotide dRpCpGpG (lane 1), was generated as
described in text and electrophoresed on 20% polyacrylamide gel next
to the markers (lane 2).
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Because oligonucleotides containing 5'-dRP may migrate differently in
polyacrylamide gels than the oligonucleotide markers used, we
engineered a marker with the same nucleotide sequence as the expected
excision product and with the dRP residue at the 5'-end. First, we
constructed an oligonucleotide duplex with a uracil residue in one
strand 4 bases upstream of the 3'-end and with a labeled phosphate
group next to it on the 5' side. This oligonucleotide duplex was then
treated with bacterial uracil-DNA glycosylase and endonuclease IV. The
combined action of these enzymes releases the 5'-deoxyribose
phosphate-containing oligonucleotide pdRpCpGpG. This product was
stabilized by reduction with NaBH4 and subjected to high
resolution electrophoresis. The pdRpCpGpG molecule almost co-migrated
with the 3-mer marker (Fig. 1c). We conclude that during
long patch BER of the U-DNA the dRP residue can be excised with at
least three nucleotides located immediately 3' to the damaged base.
Excision of dRP-oligo Depends on pol
--
The use of pol
-knockout mouse embryonic fibroblasts with a homozygous deletion in
the pol
gene allowed us to test directly whether pol
is
involved in excision steps of long patch BER. We found that wild-type
mouse cell extract released dRP-oligo as a major excision product in
long patch BER. This product was strongly reduced in the pol
null
cell extract, and the size distribution of excision products was
different (Fig. 2). To confirm that the
observed reduction in dRP-oligo release was due to the absence of pol
, the purified enzyme was added to cell extract prepared from pol
null cells (Fig. 3). As little as 2 ng of pol
could reconstitute dRP-oligo release to the level
observed in wild-type cell extract (Fig. 3, compare
lanes 4 and 6). The addition of 4 ng of pol
further stimulated dRP-oligo excision (Fig. 3, compare lanes
5 and 6).

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Fig. 2.
The release of dRP-oligo in cell
extracts. 100 ng of internally labeled U-DNA was incubated with 20 µg of extract of the wild-type or pol null cells at 32 °C for
the indicated time period as described under "Materials and
Methods." After the incubation, reaction products were reduced with
sodium borohydride and analyzed by electrophoresis on 20%
polyacrylamide gel.
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Fig. 3.
Reconstitution of dRP-oligo release in a
pol null cell extract with purified pol
. Cell extract (20 µg of protein) derived
from pol null cells was preincubated on ice for 20 min with the
indicated amounts of pol protein before the addition of U-DNA
substrate. Reactions were further incubated for 20 min at 32 °C and
processed as described above. The release of dRP-oligo by wild-type
(pol +) extract is shown in lane 6.
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Additional evidence for a role of pol
in excision was obtained by
using antibody specific to pol
. The addition of antibody blocked
the excision of dRP-oligo and resulted in an excision product pattern
characteristic for the pol
null cell extract (Fig.
4, lanes 2-4). This excision
deficiency in the presence of antibody was then corrected by addition
of pol
to the antibody-containing reaction (Fig. 4, lane
5), indicating that the effect of antibody was highly specific and
limited to blockage of pol
function. This antibody to pol
is
known to inhibit the enzyme's DNA polymerase activity (16). Based on
these experiments, we conclude that DNA repair synthesis performed by
pol
is required for dRP-oligo excision in long patch BER.

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Fig. 4.
pol dependence of
dRP-oligo release in a wild-type cell extract. Cell extract (20 µg) derived from wild-type mouse cells was preincubated on ice for 20 min with indicated amounts of pol polyclonal antibodies before the
substrate U-DNA was added. Reactions were further incubated for 20 min
at 32 °C. Lane 5 also contains 4 ng of pol in
addition to the pol antibody.
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DISCUSSION |
DNA Polymerases in Base Excision Repair--
Previous studies had
shown that pol
is the major DNA polymerase for the
single-nucleotide BER pathway (12) whereas pol
/
are thought to
be involved in PCNA-dependent long patch BER (4, 5). Recent
findings, however, suggest that pol
and pol
can substitute for
each other in long patch BER reconstituted with purified proteins (7).
The substitution of different polymerases in BER was also confirmed by
the competence of pol
null cell extracts in the in vitro
repair of both natural and reduced abasic sites in closed circular DNA
(11, 17). Thus, the biochemical proficiency of pol
and other
polymerases in both subpathways for base excision repair has been
documented. Yet, the question remained as to the DNA polymerase of
choice for the long patch BER subpathway and its precise role(s) in the
sequential mechanism. In this report we present data demonstrating that
pol
is the major DNA polymerase involved in long patch BER in
mammalian cells. Several experimental approaches used in this study
support this conclusion. First, the excision step of long patch BER is
dependent upon the pol
status of the cell extract: long patch
excision is reduced in pol
-deficient cells but can be reconstituted
by the addition of purified pol
. Second, pol
-neutralizing
antibody inhibits long patch BER excision and especially release of the dRP-oligo product. These results were unexpected because pol
has
not been proposed to participate in the long patch BER reaction. The
striking homogeneity of the excision product size (i.e. the dRP-oligo) indicates that the proteins involved in excision may predetermine the length of the excised oligonucleotide, and pol
is
a good candidate for this function. It was shown earlier that when a
suitable substrate is provided, FEN1 is able to release the AP-site
5'-sugar phosphate as part of an oligonucleotide but does not favor any
particular flap size (18). However, as we have demonstrated here in the
presence of pol
, the excision is almost strictly limited to release
of the dRP-oligo, and in pol
-deficient cell extract this particular
excision product was significantly reduced.
The Role of pol
in Coordination of BER--
It appears that
different steps of the BER reaction are coordinated and directed by
multiple interactions between the participating proteins (3). For
example, after removal of the T base, from the G-T mismatch, G-T-DNA
glycosylase remains bound to the AP site and must be displaced by AP
endonuclease (19). Further, AP endonuclease was shown to interact with
pol
, and this interaction stimulates the pol
lyase repair
reaction (20). After this step in the repair process pol
appears to
play a central role in determining the subpathway: removal of 5'-sugar
phosphate by pol
(21, 22) and interaction with DNA ligase I (23)
results in single-nucleotide BER. Alternatively, as we demonstrate
here, a pol
-dependent excision reaction may switch
repair to the long patch BER subpathway. In conclusion, whereas
multiple pathways function in BER, pol
-dependent DNA
repair operates in mammalian cells for both single-nucleotide BER and
long patch BER.