From the Department of Radiation Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111
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ABSTRACT |
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Base excision repair can proceed in either one of
two alternative pathways: a DNA polymerase -dependent
pathway and a proliferating cell nuclear antigen
(PCNA)-dependent pathway. Excision of an apurinic/apyrimidinic (AP) site by cutting the phosphate backbone on
its 3' side following incision at its 5' side by AP endonuclease is a
prerequisite to completion of these repair pathways. Using a
reconstituted system with the proteins derived from Xenopus laevis, we found that flap endonuclease 1 (FEN1) was a factor responsible for the excision of a 5'-incised AP site in the
PCNA-dependent pathway. In this pathway, DNA synthesis was
not required for the action of FEN1 in the presence of PCNA and a
replication factor C-containing fraction. The polymerase
-dependent pathway could also use FEN1 for excision of
the synthetic AP sites, which were not susceptible to
-elimination.
In this pathway, FEN1 was functional without PCNA and replication
factor C but required the DNA synthesis, which led to a flap structure
formation.
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INTRODUCTION |
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Base excision repair is a major mechanism for repair of damaged bases in DNA (1). Major lesions to be repaired by this mechanism are bases with a relatively small modification or apurinic/apyrimidinic (AP)1 sites. These types of damage are induced by ionizing radiation and exposure to various chemicals, such as alkylating agents, as well as by attack from reactive species generated during normal cellular metabolism. To protect their DNA from such persistent damage formation, most living organisms are equipped with vital mechanisms for base excision repair. In a present model for base excision repair (2), an altered base is removed by a specific DNA-N-glycosylase to leave an AP site, which is accordingly a common intermediate. The next step in this mechanism is the incision of the phosphate backbone immediately 5' to the AP site by AP endonuclease, generating a 3'-hydroxyl terminus and a 5'-terminus with a deoxyribose phosphate (dRP) group. The 5'-dRP residue is then excised, and a DNA polymerase fills in the gap. Finally, the DNA strand is sealed by the action of a DNA ligase.
Recent studies with in vitro repair systems derived from
vertebrates indicate that AP site repair in higher eukaryotes may proceed by either one of two alternative pathways: a DNA polymerase (pol
)-dependent pathway and a proliferating cell
nuclear antigen (PCNA)-dependent pathway (3, 4). The pol
-dependent pathway requires a minimum of three proteins
for AP site repair: AP endonuclease, pol
, and DNA ligase. In this
pathway, pol
catalyzes not only DNA synthesis but also excision of
a dRP residue (5). This dRP excision is via
-elimination catalyzed
by the amino-terminal 8-kDa domain of pol
. Consequently, the pol
-dependent pathway can repair an unmodified natural AP
site efficiently but not a reduced AP site or a synthetic AP site
analog, 3-hydroxy-2-hydroxymethyltetrahydrofuran (tetrahydrofuran),
neither of which is susceptible to
-elimination. In contrast, the
PCNA-dependent pathway is able to repair the synthetic AP
site analog as efficiently as the natural AP site (3). Therefore, the
excision of dRP residues during PCNA-dependent repair is
not via
-elimination but seems to be via hydrolysis. PCNA-dependent AP site repair has been reconstituted
in vitro with proteins from Xenopus laevis
ovaries consisting of three homogeneously purified proteins, AP
endonuclease, PCNA, and DNA polymerase
, and two protein fractions,
BE-1B and BE-2 (3). Although BE-1B and BE-2 have not been purified as
homogeneous proteins, the requirement of these fractions for DNA
synthesis, excision of AP sites, and ligation steps suggests that
replication factor C (RF-C), a 5'
3' exonuclease, and a DNA ligase
are included in one or the other of these fractions.
Flap endonuclease 1 (FEN1) was originally isolated as a DNA
structure-specific endonuclease that cleaves a flap strand of branched
DNA with a 5' single-stranded terminus at the position near its
junction to the double-stranded structure (6). Subsequently, it was
found to be identical or homologous to previously isolated proteins
DNaseIV (7), pL (8), 5'3' exonuclease (9), and MF1 (10). Thus FEN1
carries several distinct nuclease activities on specific structured DNA
substrates. It works as an endonuclease on 5'-flap structured DNA, as a
5'
3' exonuclease on nicked or gapped dsDNA (9, 11), and as a
ribonuclease on RNA-primed Okazaki fragments generated during
discontinuous DNA replication (10, 12). In addition, FEN1 has an
activity for removing 5'-incised AP sites (13, 14). Furthermore, recent
studies demonstrate that PCNA directly binds to FEN1 and stimulates its
activity (15, 16). Therefore, FEN1 is a candidate for the factor
responsible for AP site excision in the PCNA-dependent
repair pathway.
In this study, we cloned a cDNA coding for X. laevis
FEN1 (X-FEN1). Using the highly purified protein expressed from this cDNA, we show here that FEN1 is essential for
PCNA-dependent AP site repair and is found in the BE-2
fraction from X. laevis ovaries. FEN1 can also assist the
pol -dependent pathway in the absence of PCNA to repair
the synthetic AP site analog, which cannot be removed via
-elimination. The mode of its function in the pol
-dependent pathway is, however, distinct from that in
the PCNA-dependent pathway.
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EXPERIMENTAL PROCEDURES |
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Repair Factors-- PCNA, AP endonuclease, and the BE-2 fraction from X. laevis ovaries were prepared as described previously (3). The BE-1 fraction was prepared by the procedures described (3) except that S150 extract (17) was used as a starting material instead of the F3 fraction.
Cloning of a cDNA Coding for X-FEN1--
A part of the
X-FEN1 coding cDNA was obtained by PCR amplification of total
X. laevis oocyte cDNA with degenerative primers designed
for the conserved amino acid sequences between mouse and human FEN1
(see Fig. 1). This 240-kilo-base pair partial cDNA fragment was
used as a probe for screening an X. laevis oocyte cDNA
library in ZAP vector (18). Positive phage clones were isolated and
converted to plasmids according to the manufacturer's instructions
(Stratagene). The clone carrying the longest cDNA insert (1.9 kilobases) was chosen for further characterization. This cDNA
fragment was digested with EcoRI and either KpnI
or SalI, and the resultant, smaller fragments were subcloned
into pBS
(Stratagene). Each subclone was sequenced with a reverse primer and a T7 primer with an automated sequencer (Applied
Biosystems).
Overproduction and Purification of X-FEN1--
The coding region
of the cloned X-FEN1 cDNA was transferred into a pRSET vector for
overexpression in bacteria (19). The X-FEN1 protein was overproduced
from the pRSET carrying the X-FEN1 cDNA in a bacterial strain,
BL21(DE3)(pLysS) (Ref. 20; obtained from Novagen), by induction with
1 mM isopropyl-1-thio-
-D-galactopyranoside for 3 h at 25 °C. The bacterial cells from a 250-ml culture
were harvested by centrifugation and lysed by sonication in Buffer A
(50 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 0.1 mM EGTA, 20% glycerol, 1 mM dithiothreitol, 2 mM benzamidine-HCl, 0.2 mM phenylmethylsulfonyl fluoride) plus 500 mM NaCl. After centrifugation at
18,000 × g for 20 min, the supernatant was diluted
with 1.5 volumes of Buffer A without NaCl and loaded onto a 10-ml
column of DEAE-Sepharose FF (Amersham Pharmacia Biotech) that had been
preequilibrated with Buffer A plus 200 mM NaCl. The
flow-through fraction from the DEAE column was diluted with an equal
volume of Buffer A without NaCl and loaded onto a 1-ml Hitrap SP column
(Amersham Pharmacia Biotech) that had been preequilibrated with Buffer
A plus 100 mM NaCl. Elution was performed with a linear
gradient of 100-500 mM NaCl in Buffer A. The fractions
containing the FEN1 protein were pooled, diluted with Buffer A without
NaCl to give a conductivity equivalent to 70 mM NaCl, and
loaded onto a 1-ml column of single-stranded DNA cellulose (Sigma).
FEN1 was recovered by a stepwise elution with Buffer A plus 500 mM NaCl.
Anti-X-FEN1 Antibody-- Polyclonal antibodies against X-FEN1 were raised in a rabbit by three injections with the purified X-FEN1 protein by the standard procedures (22). Polyclonal antiserum from the rabbit was purified by using a 1-ml column of X-FEN1-affinity resin that had been prepared using Affi-Gel 10 (Bio-Rad) and the purified X-FEN1 protein (1.2 mg) according to manufacturer's instructions. Rabbit serum was diluted 10-fold in 10 mM Tris-HCl (pH 7.5) and passed 10 times through the X-FEN1-affinity column. The column was then washed with 20 ml of 10 mM Tris-HCl (pH 7.5) followed by another wash with 20 ml of 500 mM NaCl, 10 mM Tris-HCl (pH 7.5). The anti-X-FEN1 antibody was then eluted with 100 mM glycine (pH 2.5) and neutralized with 100 mM Tris-HCl (pH 8.0).
Immunoblotting of X-FEN1-- Protein samples were subjected to electrophoresis in an SDS-containing 10% polyacrylamide gel as described by Laemmli (23) and electrotransferred to an Immobilon P membrane (Millipore) in transfer buffer (10 mM 3-cyclohexylamino-1-propane sulfonic acid (pH 11.0), 10% methanol) at 100 V for 1 h in a cold room as described by LeGendre et al. (24). The blot was incubated with 1:4000 dilution of anti-X-FEN1 antibody in PBS plus 0.5% Tween 20 overnight. The immunoreactive proteins were visualized by colorimetric development after incubation with alkaline phosphatase-conjugated anti-rabbit IgG secondary antibody.
AP Site Repair Assay--
32P-Prelabeled covalently
closed circular DNA substrates containing a synthetic AP site analog,
3-hydroxy-2-hydroxymethyltetrahydrofuran (tetrahydrofuran) residue,
were prepared as described previously (3). Among these substrates, the
5'-labeled DNA has a [32P]phosphate at the position 5 nucleotides away from the lesion toward 5', whereas the 3'-labeled DNA
has a 32P atom at the position 10 nucleotides away from the
lesion toward 3'. Repair assay with these covalently closed circular
DNA substrates was conducted as described previously (3). Briefly, the
indicated proteins were incubated with either the 5'- or 3'-labeled DNA containing a tetrahydrofuran site at 25 °C for 30 min. In a standard reaction, 7 ng of X. laevis AP endonuclease, 20 ng of
X. laevis PCNA, 30 ng of X-FEN1, and 0.4 µg of protein of
the BE-1 fraction were employed. For the pol -dependent
repair with three purified proteins, 7 ng of AP endonuclease, 50 units
of X. laevis pol
, and 0.1 unit of T4 DNA ligase (Life
Technologies, Inc.) were used. After the reaction was stopped by the
addition of SDS, DNA was recovered by phenol/chloroform extraction and
ethanol precipitation, digested with either PvuII or
HinfI and subjected to electrophoresis in a denaturing 6%
(for the PvuII digests) or 20% (for the HinfI digests) polyacrylamide gel. Subsequently, the gel was subjected to
autoradiography with an x-ray film and was quantitatively analyzed with
a Fuji BAS1000 phosphorimage analyzer.
AP Site Excision Assay--
Two oligonucleotides complementary
to each other (see Fig. 3; the 3'-terminal 32P-adenosine in
the upper strand was added afterward) were prepared by an automated DNA
synthesizer in the DNA synthesis facility of Fox Chase Cancer Center. A
tetrahydrofuran site in the upper strand was introduced by using its
phosphoramidite derivative, dSpacer (Glen Research Corp.). The
prelabeled oligonucleotide substrate was prepared by annealing the two
oligonucleotides and end-filling at the 3' end of the lesion-containing
strand with [-32P]dATP and reverse transcriptase. In
the AP site excision assay, this oligonucleotide substrate (40 fmol)
was incubated with 100 ng of undamaged plasmid DNA, 7 ng of X. laevis AP endonuclease, and indicated proteins in 10 µl of the
reaction buffer containing 20 mM HEPES-KOH (pH 7.5), 100 mM KCl, 10 mM MgCl2, 1 mM dithiothreitol, 0.1 mg/ml bovine serum albumin. After
incubation at 25 °C for 30 min, the reaction mixtures were mixed
with 10 µl of sequencing gel loading solution (95% formamide, 0.5%
SDS, 0.025% bromphenol blue, 0.025% xylene cyanol FF), boiled for 1 min, and subjected to electrophoresis on a 20% polyacrylamide gel
containing 8 M urea (40 cm long) at 2400 V for 7 h.
Subsequently, the gel was subjected to autoradiography with an x-ray
film and also analyzed with a Fuji BAS1000 phosphorimage analyzer.
Detection of the PCNA/X-FEN1 Interaction-- The indicated amounts of PCNA and FEN1 were mixed in a 10 µl of buffer containing 20 mM HEPES-KOH (pH 7.5), 50 mM KCl, 0.1 mM EDTA, 20% glycerol, 1 mM dithiothreitol, 0.01% Triton X-100, and 0.1 mg/ml bovine serum albumin and incubated on ice for 20 min. Subsequently, the samples were subjected to electrophoresis in a 5% polyacrylamide gel (ratio of acrylamide to bis, 38:1) in TBE buffer (45 mM Tris borate, 1 mM EDTA). The proteins were electrotransferred to an Immobilon-P membrane and detected by immunoblotting with a monoclonal anti-PCNA antibody, 19F4 (a generous gift from Bruce Stillman), or the affinity-purified anti-FEN1 antibody as described above.
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RESULTS |
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Isolation of an X. laevis FEN1 Homolog-- To test whether FEN1 can serve for excision of a dRP residue during AP site repair by the reconstituted system with X. laevis proteins, we first isolated a cDNA coding for X. laevis FEN1. Because the amino acid sequences of human and mouse FEN1 are 96% identical (25, 26), we designed three degenerative primers from their conserved sequences and used them for nested PCR amplification on X. laevis oocyte cDNA. One of the PCR products thus obtained coded an amino acid sequence that was highly homologous to mammalian FEN1. This cDNA fragment was used as a probe for screening a cDNA library derived from X. laevis oocytes. One of the clones we isolated from the cDNA library contained an open reading frame coding for a protein, the amino acid sequence of which was more than 80% identical to mammalian FEN1 (Fig. 1). For further characterization, the protein coded by this cDNA, named X-FEN1, was overproduced in bacteria and purified as a more than 95% homogeneous protein as described under "Experimental Procedures."
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Identity of X-FEN1 as a Factor in the BE-2 Fraction Essential for
AP Site Excision--
Previous biochemical characterization of
X. laevis repair factors suggested that the BE-2 fraction
may contain a 5'3' exonuclease, the size of which should be between
29 and 66 kDa (3). To examine whether the BE-2 fraction contains the
X-FEN1 protein, we prepared a polyclonal antibody against recombinant
X-FEN1 and used it for immunoblotting. This anti-X-FEN1 antibody
recognized a protein in BE-2 that had the same mobility as that of the
recombinant X-FEN1 in SDS-containing polyacrylamide gel electrophoresis
(Fig. 2A). The same result was
obtained with another anti-X-FEN1 antibody prepared independently by D. Carroll and colleagues (data not shown). Comparison of the signal
obtained from BE-2 with those from various amounts of purified X-FEN1
revealed that the BE-2 fraction included approximately 30 ng of X-FEN1
per µl (Fig. 2A).
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Damage Excision by X-FEN1-- The recombinant X-FEN1 was tested for the activity for removing a dRP residue from the 5'-incised AP site. In this experiment, we employed a double-stranded oligonucleotide substrate carrying a tetrahydrofuran residue in the middle of one strand (Fig. 3A). In the presence of AP endonuclease, which cut the phosphate backbone at the 5' side of AP sites, X-FEN1 converted the incised product to shorter fragments (Fig. 3B, lane 3). Compared with the molecular size marker corresponding to the product that lost only the 5'-terminal tetrahydrofuran residue (Fig. 3B, lane 1), these short products appeared to result from excision of the tetrahydrofuran together with one or more 3'-adjacent nucleotides from the incised AP site. We could not detect an intermediate resulting from excision of only the sugar phosphate residue. This is consistent with the previous report that FEN1 cannot remove the 5'-terminal dRP residue as a free form but only as a part of an oligonucleotide (27).
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Analysis of Intermediate Products during FEN1-associated Repair-- To further characterize the action of FEN1 in base excision repair, we analyzed intermediate products in the repair reaction in which either the ligation or the DNA synthesis step was blocked. Ethidium bromide is known to strongly inhibit the activity of most of DNA ligases. We tested whether this DNA intercalating agent could specifically block the ligation step during AP site repair. Among the four steps in AP site repair, the incision by AP endonuclease turned out to be strongly inhibited by ethidium bromide (data not shown). When the DNA substrates had been treated with AP endonuclease prior to the addition of ethidium bromide, neither DNA synthesis nor dRP excision was significantly suppressed by the addition of 10 µg/ml ethidium bromide (Fig. 5A). However, this concentration of ethidium bromide blocked ligation and, therefore, more than 80% of the repair of tetrahydrofuran sites. Therefore, we examined the dRP excision by X-FEN1 under conditions in which the ligation step was blocked by 10 µg/ml ethidium bromide. As shown in Fig. 5B, excision of dRP residues by X-FEN1 was observed only when both PCNA and the BE-1 fraction were added together to the reaction, suggesting that X-FEN1 required PCNA and RF-C for its action on circular DNA.
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Supplementation of the Pol -dependent Repair with
X-FEN1--
As previously reported, the reconstituted system
consisting of AP endonuclease, pol
, and DNA ligase cannot repair
the tetrahydrofuran site because this lesion is not susceptible to
-elimination (5). However, the study of the reconstituted system
with X. laevis proteins (3) demonstrates that the pol
-dependent pathway can repair the tetrahydrofuran site
at a low level in the presence of the BE-2 fraction, which now turns
out to contain X-FEN1. Our recent observation also indicated that a
mouse cell extract repaired the tetrahydrofuran site by the pol
-dependent pathway (29), although not efficiently. In
these repair reactions, FEN1 may play a role in the pol
-dependent pathway to excise the tetrahydrofuran residue. Therefore, we tested whether X-FEN1 can assist the pol
-dependent pathway to repair the synthetic AP site
analog. When the 5'-labeled covalently closed circular DNA containing a
tetrahydrofuran residue was incubated with AP endonuclease, pol
,
and DNA ligase, repaired products were not detected, but some
intermediate products, resulting from an elongation by DNA synthesis of
several nucleotides, were observed (Fig.
7A, lane 2). When X-FEN1 was
added to this repair system, however, the synthetic AP site was
efficiently repaired (Fig. 7A, lane 3). Because this repair
system did not include either PCNA or RF-C, the mechanism for the FEN1
action may be different from that in the PCNA-dependent
reaction. To elucidate the PCNA-independent mechanism of FEN1 excision,
we further characterized the X-FEN1 action during the pol
-dependent repair reaction with the 3'-labeled DNA (Fig.
7B). Unlike PCNA and BE-1, which together stimulated
excision by X-FEN1 without DNA synthesis (Fig. 7B, lane 10),
pol
by itself did not stimulate the X-FEN1 activity for excision
(lane 4). However, the addition of dNTPs with pol
to the
reaction significantly induced the X-FEN1 activity (lane 8),
suggesting that the flap structure formation at the AP site by the
strand-displacing DNA synthesis was a prerequisite to its excision by
X-FEN1 in this situation. This is in stark contrast to the FEN1 action
in the PCNA-dependent pathway, in which the flap structure
was not essential for the excision.
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DISCUSSION |
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The results presented here demonstrate that FEN1 is a factor
involved in the PCNA-dependent pathway for base excision
repair. Excision of AP sites is a prerequisite to completion of base
excision repair. Typically, it proceeds via an incision of the
phosphate backbone at the 5' side of the AP site followed by another
incision at its 3' side. Whereas the 5' incision is mostly carried out through hydrolysis by AP endonuclease, the 3' incision of AP sites can
proceed via either -elimination or hydrolysis. In higher eukaryotes,
pol
has an activity for excision of a dRP residue from 5'-incised
AP site through
-elimination (5). Thus, the pol
-dependent pathway is able to complete repair of natural AP sites without additional factors for 3' incision. On the other hand,
FEN1 is able to excise a dRP residue from the 5'-incised AP site via
hydrolysis (13, 14). In this reaction, the 5'-terminal dRP residue is
released together with its 3'-adjacent nucleotide(s) as a part of an
oligonucleotide (27). The hydrolytic excision by FEN1 can be applied
not only to unmodified natural AP sites but also to modified or
synthetic AP sites that are refractory to
-elimination. These
characteristics of FEN1 confer two significant properties on the
PCNA-dependent pathway for base excision repair. First,
this pathway can repair modified or synthetic AP sites as efficiently
as unmodified natural AP sites. Second, this pathway replaces at least
two nucleotides during the repair reaction. These properties are
distinct from those of the pol
-dependent pathway, which
repairs preferentially natural AP sites through the replacement of a
single nucleotide (30, 31).
FEN1 can physically bind to PCNA with a stoichiometry of three FEN1
molecules to one PCNA trimer (16, 32). The result of our gel mobility
shift of PCNA with X-FEN1 provides additional evidence for their strong
interaction. Li et al. (15) reported that the flap
endonuclease activity of FEN1 on oligonucleotide substrates was
stimulated when a highly excess amount of PCNA was added. They also
observed a similar stimulation of FEN1 exonuclease activity by PCNA on
circular DNA but not on oligonucleotide substrates. We here
demonstrated that PCNA with the RF-C-containing fraction induced the
X-FEN1 activity for AP site excision on circular DNA but not on
oligonucleotides. The stimulation of FEN1 by PCNA on circular DNA was
detected with a reasonable molar ratio for their stoichiometric
interaction. The simplest model provided from our result and the
established function of PCNA and RF-C is that FEN1 bound to PCNA is
recruited to the nicked site by RF-C. Because we employed the BE-1
fraction instead of the purified RF-C, it is still possible that other
factors in BE-1, such as DNA polymerase or DNA ligase, may also
participate in the stimulation of X-FEN1. In previous studies with an
S150 extract from X. laevis oocytes, we observed that
omission of dNTPs from the repair reaction blocked not only DNA
synthesis but also excision of the 5'-terminal AP site (33). This
result appears to contradict the result described here. One possible
explanation to resolve this contradiction is that DNA polymerase
(or
) may block the nuclease activity of FEN1 in the absence of DNA
synthesis in the S150 extract, whereas an excess amount of FEN1 in the
reconstituted system may overcome the blocking by DNA polymerase. It
remains to be determined whether PCNA can interact with DNA polymerase
/
and FEN1 simultaneously and, if so, whether the 5'-terminus of
the nicked site can be accessible to FEN1 in this complex.
The activity for AP site excision by X-FEN1 was also stimulated by pol
. Because it was observed only when DNA synthesis was allowed, the
stimulation by pol
seems to target the flap endonuclease activity.
Li et al. (15) and Wu et al. (16) reported that
the flap endonuclease of FEN1 can be stimulated by PCNA. In our
experiments, however, the efficiency of AP site excision by X-FEN1 with
pol
was comparable to that by X-FEN1 with PCNA and the BE-1
fraction, suggesting that PCNA is not essential for the FEN1 activity
on the flap-structured DNA formed by pol
. These observations may be
explained by the possibility that a flap-structured DNA can be a better
substrate for FEN1 than a nicked DNA and accordingly can be processed
at an apparently efficient rate without PCNA. Indeed, it has been
reported that FEN1 has a 2-4-fold higher affinity for the flap
structure than the nicked site (11). Another possibility is that pol
may play the role of PCNA in stimulation of FEN1 on a
flap-structured DNA. However, this possibility was ruled out by our
observation that once DNA synthesis displaced several nucleotides of
the downstream strand carrying a 5'-incised AP site, X-FEN1 was equally
active for excision in either the presence or the absence of pol
(data not shown). Thus, the generation of a 5'-flap structure is
sufficient to explain the stimulation of FEN1 by pol
.
The efficient excision of AP sites by FEN1 with pol allowed the pol
-dependent pathway to repair tetrahydrofuran sites that
cannot be excised by dRP lyase activity of pol
. Recently, Klungland
and Lindahl (34) reported that FEN1 is involved in base excision repair
for the AP sites reduced with sodium borohydride and the oxidized AP
sites generated by ionizing radiation. These lesions are similar to the
tetrahydrofuran sites used in this study for their lack of
susceptibility to
-elimination. However, the results described here
do not support their model, in which the pol
-dependent
repair assisted by PCNA-stimulated FEN1 may be a major contributor to
the PCNA-dependent pathway in base excision repair.
Furthermore, it was observed that a pol
-knockout cell extract
repaired the tetrahydrofuran site as efficiently as a pol
-proficient isogenic cell extract (29). This observation clearly
indicates the participation of a DNA polymerase other than pol
,
most likely pol
or
, in the repair of modified AP sites.
Eukaryotes have other structure-specific nucleases and 5'3'
exonucleases in addition to FEN1. These enzymes may also serve for
excision of altered AP sites. XPG is a protein coded by the gene
responsible for the xeroderma pigmentosum G phenotype and is required
for nucleotide excision repair. It is also homologous to FEN1 in three
domains and has a structure-specific endonuclease activity that incises
single-stranded DNA at its 3' border to double-stranded DNA (25, 35).
When it is substituted for FEN1, however, XPG cannot support repair of
altered AP sites (34).2
However, the possibility is not ruled out that some other factor(s) that can interact with XPG may be required for excision of AP sites by
XPG. DNaseV has both 5'
3' and 3'
5' exonuclease activities and
forms a complex with pol
(36). It is reported that DNaseV enhances
the repair synthesis by pol
at the nicked site generated by AP
endonuclease (37). Although this enzyme has not been well characterized
yet, DNaseV may be a nuclease to excise altered AP sites in the pol
-dependent pathway. It has not been determined whether
DNaseV can interact with PCNA. Thus, it remains to be examined whether
FEN1 is the only factor responsible for the excision step in the
PCNA-dependent base excision repair.
Yeast FEN1 homologs (the RAD27 gene product in Saccharomyces cerevisiae and the rad2 gene product in Schizosaccharomyces pombe) have been characterized for their cellular functions. The yeast cells lacking the functional RAD27 or rad2 gene exhibit various phenotypes, such as moderate sensitivity to UV radiation, hypersensitivity to methylmethane sulfonate, deficient chromosome segregation, conditional lethality, and accumulation of S phase cells (26, 38-40). These phenotypes suggest involvement of FEN1 homologs in DNA replication, repair, and cell cycle control. Among these phenotypes, the hypersensitivity to the alkylating agent particularly supports our conclusion that FEN1 is one of the factors employed in base excision repair. Recently, Tishkoff et al. (41) reported that a mutation in RAD27 resulted in a strong mutator phenotype that caused duplication of sequences ranging from 5 to 108 base pairs flanked by direct repeats of 3-12 base pairs. These authors proposed that this mutagenic process may be initiated at DNA replication on the lagging strand, where the RAD27 gene product is required for legitimate joining of Okazaki fragments. In the RAD27-deficient cells, a displaced 5'-flap may not be removed efficiently, leading to a high incidence of duplication between distant short direct repeats (Fig. 8A). Our results provide an additional model for generation of the same type of mutations, which can be initiated during base excision repair in RAD27-deficient cells (Fig. 8B). It is likely that the duplications generated during base excision repair may be relatively short compared with those arising from DNA replication because, unlike the replication machinery, the base excision repair complex may lack some factors, such as a helicase and a single-strand DNA-binding protein, that are required for displacing a long range of the downstream DNA strand. This model can be tested by examining whether treatment with the alkylating agent can increase the specific type of mutations in these cells.
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ACKNOWLEDGEMENTS |
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We thank Gordon Chan for providing the X. laevis total cDNA; M. Bibikova and D. Carroll for providing their anti-X. laevis FEN1 antibody; Bruce Stillman for the anti-PCNA antibody; R. D. Wood for the purified XPG protein; and A. Bellacosa, S. W. Johnson, R. A. Katz, and A. T. Yeung for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants CA63154 and CA06927 and by an appropriation from the Commonwealth of Pennsylvania.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF036327.
To whom correspondence should be addressed: Fox Chase Cancer
Center, 7701 Burholme Ave., Philadelphia, PA 19111. Tel.:
215-728-5272; Fax: 215-728-4333; E-mail: y_matsumoto{at}fccc.edu.
1 The abbreviations used are: AP, apurinic/apyrimidinic; FEN1, flap endonuclease 1; PCNA, proliferating cell nuclear antigen; RF-C, replication factor C; pol, DNA polymerase; dRP, deoxyribose phosphate; PCR, polymerase chain reaction.
2 K. Kim and Y. Matsumoto, unpublished data.
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REFERENCES |
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