From the Life Sciences Division, M888, Los Alamos
National Laboratory, Los Alamos, New Mexico 87545 and the
¶ Department of Radiation Oncology, Fox Chase Cancer
Center, Philadelphia, Pennsylvania 19111
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ABSTRACT |
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There are two distinct pathways for the removal
of modified DNA bases through base excision repair (BER) in
vertebrates. Following 5' incision by AP endonuclease, the pathways
diverge as two different excision mechanisms are possible. In
short-patch repair, DNA polymerase Damage to DNA bases can occur spontaneously under physiological
conditions. Bases may be modified by deamination, or lost entirely due
to hydrolysis of the N-glycosidic bond that links them to
the sugar-phosphate backbone. Reactive oxygen species that are the
by-products of normal cellular respiration also damage DNA. Exogenous
agents such as chemical mutagens and ionizing radiation are additional
sources of base modification. Aberrant DNA bases generated by such
processes can be restored by base excision repair (BER)1 (reviewed in Refs. 1
and 2). This repair system includes several types of DNA glycosylases
that recognize and remove many types of modified bases to leave an
apurinic or apyrimidinic site (AP site). Alternatively, an AP site may
be the direct result of damage. The AP site must be removed during the
course of repair. AP endonuclease initiates the process by hydrolyzing
the phosphodiester bond immediately to the 5' side of the abasic site.
This enzyme is capable of recognizing various classes of abasic lesions
(3). After this incision has been made, there are two mechanistically distinct methods for AP site removal that distinguish short-patch BER
from long-patch BER in higher eukaryotes. Short-patch BER is a DNA
polymerase In long-patch BER, the repair patch size is typically 2-8 nucleotides
in length. This pathway also utilizes AP endonuclease for 5'-incision,
but the AP site is not removed by DNA polymerase PCNA and FEN-1 are capable of direct physical interaction, as first
identified by yeast two-hybrid screening (13) and confirmed by in
vitro binding assays (13-18). Furthermore, PCNA can stimulate FEN-1 nuclease activity (13, 14, 18). Both proteins participate in DNA
replication and long-patch BER, and thus it is likely that these
proteins may co-exist within a macromolecular assembly during either of
these processes. Because PCNA and FEN-1 have a proven capacity for
interaction, it is tempting to suppose that direct physical interaction
of these proteins occurs during one or more steps of replication or
repair. The most direct test of this hypothesis is to selectively
abolish the ability of PCNA and FEN-1 to interact with one another, and
then to determine whether loss of this function affects replication or
repair efficiency. We have used this strategy to investigate the
potential role of this interaction in long-patch BER. In this report,
we show that the PCNA binding activity of FEN-1 is important for
excision in BER, thus establishing a specific repair function for the
interaction of these two proteins.
Bacterial Expression Plasmids--
The wild type human PCNA
expression plasmid has been described previously (17). The L126D/I128E
double point mutant derivative of this plasmid was generated by
site-directed mutagenesis using the QuickChange Mutagenesis protocol
(Stratagene, La Jolla, CA) with the high-fidelity Pfu DNA
polymerase and mutagenic oligonucleotides 5'-GGATTTAGATGTTGAACAGGATGGAGAACCAGAACAG-3' and
5'-CTGTTCTGGTTCTCCATCCTGTTCAACATCTAAATCC-3'. This primer pair created
an FokI restriction site to facilitate screening. The
plasmid pET-FCH to express wild type human FEN-1 with a 6-histidine
C-terminal purification tag has been described (19-21). This plasmid
was modified by site-directed mutagenesis to create the F343A/F344A
mutant FEN-1 expression plasmid by QuickChange mutagenesis using
oligonucleotides 5'-GGCCGCCTGGATGATGCCGCCAAAGTGACCGGCTCACTC-3' and
5'-GAGTGAGCCGGTCACTTTGGCGGCATCATCCAGGCGGCC-3'. This primer pair was
designed to destroy a naturally occurring BstEII restriction site in order to facilitate screening. The expression constructs were
verified by DNA sequencing. In both the pET-FCH wild type FEN-1
parental vector and the F343A/F344A mutant derivative, a single
nucleotide disagreement with the published human FEN-1 sequence (22)
was observed. Nucleotide 247 of the FEN-1 open reading frame was
"C" rather than "T" as found in the originally published DNA
sequence. The resultant CAT codon specifies a histidine 83 residue in
both of the proteins (wild type and mutant FEN-1) used in the present
study, in place of tyrosine-83 obtained by conceptual translation of
the published DNA sequence. The C-247 nucleotide was present in the
original plasmid prepared by direct subcloning of the
NcoI-BamHI restriction fragment from amplified FEN-1 cDNA (20), raising the possibility that there may be
polymorphism at this position. Supporting this possibility, histidine
in place of tyrosine is a very conservative amino acid substitution; at an evolutionary distance of 2 PAM (percentage of
accepted point mutations), only phenylalanine
and tryptophan substitutions are tolerated more frequently (23). In any
case, the histidine substitution had no effect on the catalytic
activity of FEN-1, based on comparison of enzymatic specific activity
with tyrosine 83 wild type FEN-1 created with mutagenic
oligonucleotides
5'-AACGGCATCAAGCCCGTGTACGTATTTGATGGCAAGCCGCCA-3' and
5'-TGGCGGCTTGCCATCAAATACGTACACGGGCTTGATGCCGTT-3' (data not shown). This primer pair created a SnaBI restriction site to
facilitate screening.
Affinity Interaction Binding Assay--
Escherichia
coli strain BL21(DE3) cells harboring either wild type or
mutant PCNA expression plasmid, wild type or mutant FEN-1
plasmid, or pET28b vector (Novagen, Madison, WI) without cDNA
insert were grown to a density of approximately 0.6 absorbance units at
600 nm, then 0.8 mM
isopropyl- Protein Purification--
His6-tagged wild type and
mutant FEN-1 were purified with HisBind affinity resin (Novagen)
according to the manufacturer's instructions. Wild type and mutant
PCNA were purified by a method previously described for wild type PCNA
(17). AP endonuclease and the BE-1 fraction from Xenopus
laevis ovaries were prepared as described previously (7, 8). The
BE-1 fraction contains pol DNA Polymerase AP Site Repair Assay--
32P-Prelabeled covalently
closed circular DNA substrates containing a synthetic AP site analog,
the 3-hydroxy-2-hydroxymethyltetrahydrofuran residue, were prepared as
described previously (8). Among these substrates, the 5'-labeled DNA
has a 32P at the position 5 nucleotides away from the
lesion toward 5' while the 3'-labeled DNA has a 32P at the
position 10 nucleotides away from the lesion toward 3'. Repair assays
with these DNA substrates were conducted as described previously (8).
Briefly, indicated proteins were incubated with either the 5'- or
3'-labeled DNA containing a 3-hydroxy-2-hydroxymethyltetrahydrofuran site for 30 min at 25 °C in the presence of 20 µM
dATP, 20 µM dCTP, 20 µM dGTP, 20 µM TTP, and 2 mM ATP. In a standard reaction, 7 ng of X. laevis AP endonuclease, 10 ng of human PCNA, 10 ng of human FEN-1, and 0.4 µg of protein of the X. laevis
BE-1 fraction were used. For the pol Flap Endonuclease Assay--
The enzymatic activity of FEN-1 was
assayed as described previously (20). Briefly, a three-oligonucleotide
5'-flap substrate was assembled by annealing a 3'-biotinylated 31-mer
template strand, a 5'-fluoresceinated 34-mer flap strand (14 nucleotides annealed and 20 nucleotides displaced), and a 16-mer
upstream annealed primer strand. The 5'-fluoresceinated flap DNA was
attached to streptavidin-coated microspheres, and 50-100
pM substrate was mixed with enzyme in 500 µl of 50 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 100 µg/ml bovine serum albumin at room temperature. Bead-associated fluorescence was measured in real time from 0 to 500 s by flow cytometry.
Exonuclease Assay--
Three synthetic oligonucleotides were
annealed to build a nicked DNA substrate for measurement of exonuclease
activity. A 57-mer template strand
(5'-TCGAGTTATTTAAACCATGCATCTAATGTTTTTTGCTTAGTTTTGTTTGCAAGCTTG-3') was
annealed to a perfectly complementary 36-mer primer
(5'-AGCAAAAAACATTAGATGCATGGTTTAAATAACTCG-3'). The
resulting duplex DNA possessed a 1-nucleotide gap at the 3'-end of the
36-mer that was used as a labeling site for incorporation of
[ Characterization of the I126D/L128E Mutant of PCNA--
The
crystal structure of p21 peptide complexed with human PCNA has been
reported (24). FEN-1 interacts with PCNA by a mechanism analogous to
that of p21 (16, 17), so the PCNA-p21 structural information was used
to design PCNA mutants predicted to exhibit FEN-1-binding defects. A
series of mutations was introduced into PCNA, and the mutant proteins
were tested for their ability to bind to
FEN-1.2 The I126D/L128E
double point mutant of PCNA was identified as having a particularly
severe defect in FEN-1 binding activity. This mutant of PCNA was
purified and characterized (Fig. 1) for use as a tool to investigate the role of the PCNA-FEN-1 interaction in
long-patch BER.
Bacterially expressed wild type and I126D/L128E mutant human PCNA were
purified to apparent homogeneity (Fig. 1A). The PCNA subunit
migrates with an apparent molecular mass of 34 kDa on a denaturing
SDS-PAGE gel, although its calculated molecular mass is 29 kDa. An
affinity interaction assay was used to compare the FEN-1 binding
activities of wild type and mutant PCNA. Lysates from bacteria
expressing polyhistidine (His6)-tagged FEN-1 and either
wild type or mutant PCNA were mixed, and nickel-charged metal chelate
resin was added to capture the His6-tagged FEN-1 along with
any associated PCNA. As expected, wild type PCNA bound avidly to FEN-1
(Fig. 1B, lane 1). In contrast, the I126D/L128E mutant of
PCNA displayed only a background level of nonspecific binding (Fig.
1B, lane 2), similar to that observed in negative control
assays in which FEN-1 was omitted (Fig. 1B, lanes 3 and 4). Thus, the I126D/L128E mutation virtually abolished FEN-1
binding activity. Nonetheless, both wild type and mutant PCNA formed
the characteristic 87-kDa homotrimeric species when analyzed by gel filtration chromatography, confirming that intersubunit interaction was
unaffected by the site-directed mutagenesis. On a Sephacryl S-300 HR
16 × 600 mm column, the parameter K, which is given by: (elution
volume)
The I126D/L128E mutant of PCNA was, however, severely defective in the
ability to stimulate DNA synthesis by DNA polymerase Base Excision Repair Activities of Wild Type and Mutant
PCNA--
The 3-hydroxy-2-hydroxymethyltetrahydrofuran abasic site
analog can be completely repaired by long-patch BER in the presence of
dNTPs, AP endonuclease, FEN-1, PCNA, and a protein fraction partially
purified through four chromatographic steps that has been designated
BE-1 (7, 8). BE-1 provides RF-C, DNA polymerase
The 5'-32P-labeled substrate is most useful for the
observation of repair synthesis. Because removal of the synthetic AP
site requires a second cleavage event to the 3' side of the initial incision, 3'-32P-labeled substrate must be used to analyze
excision. AP endonuclease treatment produced a 3'-labeled incised
fragment with a 5'-terminal AP site (Fig. 2B, lane 1).
Although this AP end cannot be ligated, DNA ligase can covalently
attach the AMP moiety from ATP to the 5'-terminal AP site via a
pyrophosphoryl linkage to form a species referred to as a ligation
intermediate (7), by analogy with the intermediate generated during
normal ligation of base-containing ends. Addition of BE-1, which
contains DNA ligase, converted most of the incised fragment to ligation
intermediate (Fig. 2B, lane 2). Despite the presence of
FEN-1 nuclease in this reaction, little if any excision was observed.
However, the inclusion of wild type PCNA greatly stimulated excision by
FEN-1 (Fig. 2B, lane 3). Excision was evident as the
conversion of ligation intermediate to smaller product and as the
appearance of an excised band that corresponds in size to the removal
of the 5'-terminal AP site plus one additional nucleotide
(characterized in Ref. 7). It is likely that some of the excised
product became modified by DNA ligase to generate a novel post-excision
ligation intermediate that co-migrates with the incised product; this
explains the apparent increase in the intensity of the incised band
upon activation of excision (compare lanes 2 and
3 of Fig. 2B). Minor excision products
corresponding to removal of successive additional nucleotides are
sometimes seen in this assay system (7). The inclusion of the
I126D/L128E mutant of PCNA stimulated the production of only a small
amount of excised product (Fig. 2B, lane 4). The slight
stimulation of excision observed with the mutant PCNA may indicate that
interaction with FEN-1, while dramatically reduced, is not completely
abolished by this mutation. Consistent with the results shown in Fig.
2A, wild type but not mutant PCNA supported complete repair
in the fully reconstituted reaction containing all components (Fig.
2B, lanes 6 and 7).
The I126D/L128E mutant of PCNA, unable to interact with DNA polymerase
Characterization of the F343A/F344A Mutant of FEN-1--
The
His6-tagged wild type human FEN-1 and its F343A/F344A
derivative were purified to near homogeneity by metal chelate affinity chromatography (Fig. 3A). The
F343A/F344A mutant of FEN-1 was severely defective in binding to PCNA.
In the affinity interaction assay, wild type FEN-1 bound well to PCNA,
whereas mutant FEN-1 showed little if any specific binding to PCNA
(Fig. 3B). However, the catalytic behavior of the
F343A/F344A mutant of FEN-1 was identical to that of the wild type
enzyme as judged by several criteria. The enzyme concentration
dependence of 5'-flap DNA substrate cleavage was measured in real time
using a flow cytometry-based endonuclease assay (Fig.
4) as described previously (20). The apparent first-order rate constant (kobs) was
estimated from the half-time of the reaction using the formula
kobs = ln2/(t1/2) and plotted
as a function of enzyme concentration. Although the reaction mechanism
is likely to be more complex, the data can be interpreted in terms of a
two-step mechanism consisting of a binding step and a cleavage step
(20, 21). At low enzyme concentrations, the reaction kinetics are
dominated by the binding step and are thus sensitive to enzyme
concentration. At high enzyme concentrations, the binding step becomes
very fast and the reaction kinetics become limited by the cleavage
rate. Thus, the plot of kobs versus
[enzyme] is similar to a binding curve, and the data can be described
well by fitting to a single site binding curve which has the shape of a
hyperbola (y = (a)(x)/(b + x), where y = kobs, Base Excision Repair Activities of Wild Type and Mutant
FEN-1--
Wild type and F343A/F344A mutant FEN-1 were combined with
other BER reaction components to repair the 3'-32P-labeled
substrate (Fig. 6). Each reaction
contained the standard amount of AP endonuclease, PCNA, and protein
fraction BE-1 (RF-C, DNA polymerase
With 1 ng/reaction wild type FEN-1, more than 80% of the DNA was
repaired in the complete reaction (Fig. 6, right panel), yet, in the absence of dNTPs, only about 50% excision was observed at
the same FEN-1 concentration (Fig. 6, left panel). However, the intensity of the signal in the excised band underestimates the
total amount of excision that has taken place, because the excised
fragment can undergo 5'-phosphoryl-AMP modification by DNA ligase to
generate a type of post-excision ligation intermediate that co-migrates
with the incised band (evidence for this was discussed for Fig.
2B). Thus, there was a very good quantitative correlation
between excision and repair for wild type FEN-1. This was not true for
the mutant FEN-1, however. Mutant FEN-1 failed to produce significant
excision in the absence of dNTPs, even at 10 ng/reaction (data not
shown). These results suggest that wild type and mutant FEN-1 employ
different excision mechanisms during PCNA-dependent BER
(see "Discussion" for details).
Wild Type and Mutant FEN-1 Behave Similarly in a DNA Polymerase
The addition of PCNA did not stimulate nor inhibit FEN-1-mediated
excision in this system. Two distinct modes of PCNA-FEN-1 interaction
can be envisioned. PCNA and FEN-1 can form a complex that is free of
DNA, or the DNA-bound form of PCNA can bind to FEN-1. In studies that
use short oligonucleotide substrates (6), it is not clear which mode of
interaction may be applicable, because PCNA can bind to DNA with free
ends to establish an equilibrium between DNA-bound and DNA-free PCNA.
The PCNA loading factor RF-C is not required for this type of DNA
binding. In Fig. 7B, a closed circular DNA substrate was
used in the absence of RF-C, so DNA-bound PCNA was precluded. PCNA not
bound to DNA was unable to stimulate FEN-1 nuclease activity. Given
this result, one might expect that PCNA could be inhibitory in this
system, if nonproductive DNA-free PCNA-FEN-1 complexes are formed.
However, at 0.1 ng/reaction of FEN-1 (the half-maximal concentration),
a nearly 50-fold molar excess of PCNA trimer (i.e. 10 ng/reaction) had no effect on excision.
FEN-1 is a structure-specific endonuclease and 5'-3' exonuclease
that is involved in Okazaki fragment processing during lagging strand
synthesis of DNA replication. Its role in BER was first suggested by
genetic experiments in Saccharomyces cerevisiae. Strains
lacking RAD27, the yeast homolog of FEN-1, showed increased sensitivity to methyl methanesulfonate, a DNA alkylating agent that
generates BER-repairable lesions (28, 29). Recent work has confirmed
the requirement for FEN-1 in long-patch BER in vertebrates (6, 7). The
DNA-binding protein PCNA has long been recognized as an essential
co-factor of the replication and repair DNA polymerases The removal of the AP site lesion by excision appears to be a
rate-limiting step in BER when the concentration of FEN-1 is not in
excess. PCNA and FEN-1 interaction domains were disrupted by
mutagenesis, and the non-interacting PCNA and FEN-1 mutant proteins
each exhibited excision defects (Figs. 2 and 6). At 0.1, 0.3, and 1.0 ng/reaction, mutant FEN-1 was defective in both excision and overall
repair as compared with wild type FEN-1 (Fig. 6). Wild type FEN-1 was
about 10-fold more potent in reconstituting repair activity than the
mutant FEN-1 that was unable to bind to PCNA. These results demonstrate
that direct physical interaction of PCNA and FEN-1 is required for
maximum repair efficiency. Thus, physical contact between these
proteins appears to be a feature of the BER excision mechanism.
FEN-1 can act as a 5'-3' exonuclease at nicks, and as a
structure-specific endonuclease to cleave the unannealed strand of branched structures such as the 5'-flap or pseudo Y. FEN-1 is unable to
remove a 5'-terminal AP site as a free sugar phosphate, but it can
cleave farther down the strand to excise the AP site as part of a short
oligonucleotide, predominantly a dinucleotide (7, 36-38). This
property of FEN-1 activity helps to justify in mechanistic terms the
clear distinction between short- and long-patch BER; the 1 nucleotide
repair patch size in short-patch BER arises because the DNA polymerase
In the PCNA-dependent BER system, is FEN-1-mediated
excision the result of exo- or endonuclease-type activity? In the
absence of dNTPs, strand-displacing DNA synthesis cannot proceed, so
only an exonuclease-type substrate can be available for excision in such assays. Under these conditions, excision by wild type FEN-1 was
well correlated with overall repair observed in parallel reactions containing dNTPs (Fig. 6; as discussed under "Results," the
quantitation of the excised band intensity that is plotted
underestimates the total extent of excision, because there is another
post-excision species that co-migrates with the incised band). Thus,
the exonuclease-type excision activity of wild type FEN-1 (Fig. 6,
left panel) was adequate to account for most if not all of
the excision that was required during complete repair (Fig. 6,
right panel). This was not the case for mutant FEN-1,
however. Under exo-specific conditions, no appreciable excision
activity was observed for mutant FEN-1 at 1 ng/reaction (Fig. 6,
lane 11) or 10 ng/reaction (data not shown). However, 1 ng/reaction mutant FEN-1 supported 60% repair (Fig. 6, lane
22) under conditions in which strand-displacing DNA synthesis was
possible. Because excision is a prerequisite for complete repair, these
data strongly suggest that mutant FEN-1 employed endonuclease-type
excision activity during BER. Consistent with this, under the
endo-specific conditions of the DNA polymerase In reconstituted systems using purified proteins, either DNA polymerase
The short-patch BER pathway is mechanistically well established. An AP
site attracts AP endonuclease, which makes the 5'-incision. AP
endonuclease physically interacts with DNA polymerase accounts for both excision
activity and single nucleotide repair synthesis. In long-patch repair,
the damage-containing strand is excised by the structure-specific
endonuclease FEN-1 and approximately 2-8 nucleotides are incorporated
by proliferating cell nuclear antigen (PCNA)-dependent
synthesis. PCNA is an accessory factor of DNA polymerases
and
that is required for DNA replication and repair. PCNA binds to FEN-1
and stimulates its nuclease activity, but the physiological
significance of this interaction is unknown. The importance of the
PCNA-FEN-1 interaction in BER was investigated. In a reconstituted BER
assay system containing FEN-1, omission of PCNA caused the accumulation
of pre-excision reaction intermediates which could be converted to
completely repaired product by addition of PCNA. When dNTPs were
omitted from the reaction to suppress repair synthesis, PCNA was
required for the formation of excised reaction intermediates. In
contrast, a PCNA mutant that could not bind to FEN-1 was unable to
stimulate excision. To further study this effect, a mutant of FEN-1 was
identified that retained full nuclease activity but was specifically
defective in binding to PCNA. The mutant FEN-1 exhibited one-tenth the
specific activity of wild type FEN-1 in the reconstituted BER assay,
and this repair defect was due to a kinetic block at the excision step
as evidenced by the accumulation of pre-excision intermediates when
dNTPs were omitted. These results indicate that PCNA facilitates
excision during long-patch BER through its interaction with FEN-1.
INTRODUCTION
Top
Abstract
Introduction
References
-dependent pathway that requires an unaltered deoxyribose phosphate (dRP) sugar moiety as the AP site. The
5'-terminal dRP resulting from AP endonuclease incision is removed in a
-elimination reaction catalyzed by DNA polymerase
, leaving a
1-nucleotide gap (4). Single nucleotide repair synthesis by DNA
polymerase
fills the gap, and the nick is sealed by the DNA ligase
III/XRCC1 heterodimer or other ligase to complete repair.
. Instead, the flap
endonuclease/5'-3' exonuclease FEN-1 (reviewed in Ref. 5) removes the
5'-terminal dRP moiety along with at least one adjacent nucleotide to
leave a gap of two or more nucleotides (6, 7). Because it relies on
phosphodiester bond hydrolysis for excision, long-patch BER can repair
regular AP sites and also altered AP sites that are not susceptible to
-elimination. Sites with oxidized or reduced sugar groups and those
with fragmented bases or sugars can be repaired only by long-patch BER.
This versatility may be especially important in the repair of DNA
damage caused by ionizing radiation. For example, about 80% of the
-irradiation-induced DNA lesions that can be repaired by BER are
resistant to dRP elimination by DNA polymerase
and require FEN-1
for processing (6). The investigation of long-patch BER in cell
extracts and reconstituted assay systems has been aided by the
availability of synthetic AP site analogs such as
3-hydroxy-2-hydroxymethyltetrahydrofuran that cannot be repaired by
short-patch BER. Repair of such sites requires proliferating cell
nuclear antigen (PCNA) (Refs. 8 and 9), a toroidal homotrimeric
DNA-binding protein that encircles template DNA. PCNA forms a
holoenzyme complex with DNA polymerases
or
in conjunction with
replication factor C (RF-C), the PCNA loading factor (reviewed in Ref.
10). PCNA dependence in long-patch BER has also been demonstrated using
a regular AP site as substrate and selectively observing repair patches
of greater than 1 nucleotide in length by exploiting strategically
placed restriction sites (11) or position-specific deoxyribonucleotide
incorporation (12). These results suggest a model in which AP
endonuclease, FEN-1, PCNA, RF-C, a PCNA-dependent DNA
polymerase, probably DNA polymerase
, and DNA ligase are used for
long-patch BER. Complete repair can be achieved with these proteins
(6-8), and cell extracts lacking DNA polymerase
are fully capable
of repair (9, 12). However, in some assay systems, DNA polymerase
can substitute for the PCNA-DNA polymerase
holoenzyme complex in
FEN-1-dependent BER (6).
EXPERIMENTAL PROCEDURES
-D-thiogalactopyranoside was added to induce
protein expression and growth was continued for an additional 3 h
at 37 °C. Cells were harvested by centrifugation and cell pellets
were resuspended in 50 mM Tris-HCl, 150 mM
NaCl, 0.2 mg/ml lysozyme, 1 mM benzamidine, 0.5 mM phenylmethylsulfonyl fluoride, 10 µM
pepstatin A, 10 µg/ml chymostatin, 10 µg/ml aprotinin, pH 7.4, at a
ratio of 1 ml/35-ml culture and briefly sonicated. The cell lysates
were clarified by two cycles of centrifugation at 16,000 × g. Binding assay mixtures consisted of 70 µl of 50% NiSO4-charged iminodiacetic acid metal chelating Sepharose
resin (Pharmacia Biotech, Piscataway, NJ), 150 µl of lysate from
cells expressing His6-tagged wild type or mutant FEN-1, or
negative control lysate from pET28b vector-containing cells, 150 µl
of lysate from cells expressing untagged wild type or mutant PCNA, and
150 µl of 50 mM Tris-HCl, 150 mM NaCl, pH
7.4, added to increase mixing efficiency. Mixtures were incubated for
90 min at 4 °C with continuous gentle rocking, then washed six times
with 0.8 ml of 50 mM Tris-HCl, 150 mM NaCl, 60 mM imidazole, pH 7.4. Protein complexes were eluted by
heating to 100 °C with 80 µl of 2 × Laemmli SDS gel sample
buffer, and analyzed on 12% gels by SDS-polyacrylamide gel
electrophoresis (PAGE) and Coomassie Blue staining.
, RF-C, and DNA ligase activities. Rat
DNA polymerase
was overexpressed in bacteria and purified as
described previously (4).
Stimulation Assay--
Stimulation of the
activity of calf thymus DNA polymerase
(a generous gift from Drs.
Chen-Keat Tan and Antero G. So of the University of Miami) by PCNA
proteins was measured as described previously (8). Briefly, each assay
contained 0.5 µg of substrate consisting of poly(dA) template and
oligo(dT) primer at 5:1 molar ratio in 50 mM bis-Tris-HCl,
10 mM KCl, 6 mM MgCl2, 0.4 mg/ml bovine serum albumin, 1 mM dithiothreitol, 50 µM [
-32P]TTP, pH 6.5. One unit of
polymerase activity corresponds to the incorporation of 1 pmol of TMP
into acid-precipitable material in 30 min at 37 °C.
-dependent repair
reactions, 7 ng of AP endonuclease, 10 ng of FEN-1, and 50 ng of rat
pol
were used. After the reaction was stopped by the addition of
SDS, DNA was recovered by phenol/chloroform extraction and ethanol
precipitation, digested with HinfI and subjected to
electrophoresis in a denaturing 20% polyacrylamide gel. Subsequently,
the gel was subjected to autoradiography with an x-ray film and was
quantitatively analyzed with a Fuji BAS1000 phosphorimage analyzer.
-32P]dATP. A 100-µl reaction containing 12 µM duplex and 10 units of Sequenase version 2.0 DNA
polymerase (U. S. Biochemical, Cleveland, OH) in 25 mM
HEPES, pH 7.4, 10 mM MgCl2, 2 mM
dithiothreitol, 50 µg/ml bovine serum albumin, 50 µM
dATP, 20 µCi of [
-32P]dATP was incubated for 10 min
at 37 °C to generate a 3'-end 32P-labeled 37-mer strand.
The DNA polymerase was inactivated by incubation for 10 min at
70 °C, then unincorporated dATP was separated from the labeled
duplex using a 0.8-ml Sephadex G-50 spin column. A 2-fold molar excess
of the perfectly complementary upstream primer 20-mer
(5'-CAAGCTTGCAAACAAAACTA-3') was annealed to the labeled 37-mer/57-mer
duplex to generate an internally nicked substrate with blunt ends.
Exonuclease activity was assayed in a 20-µl reaction volume
containing 300 nM 32P-labeled DNA substrate and
0, 1, 2, or 4 µM wild type or F343A/F344A mutant FEN-1 in
a buffer of 28 mM imidazole, 12.5 mM HEPES, 0.5 mM Tris-HCl, 14 mM NaCl, 5% glycerol, 5 mM MgCl2, 1 mM dithiothreitol, 25 µg/ml bovine serum albumin, pH 7.4. Samples were incubated for 60 min
at 32 °C, then reactions were terminated by the addition of 20 µl
of 94% formamide, 10 mM EDTA, 0.1% xylene cyanol, 0.1% bromphenol blue and heated to 90 °C for 10 min. 15 µl of this mixture (approximately 10,000 cpm per lane) was run on a 15%
polyacrylamide (19:1 acrylamide:bis-acrylamide), 7 M urea,
10 mM Tris-HCl, 1 mM EDTA, pH 8.0, denaturing
gel at 65 watts constant power for 2 h. The gel was dried and
subjected to autoradiography (3 days exposure to Kodax BioMax film) or
quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Substrate remaining was calculated as: 37-mer area intensity/total
integrated area intensity divided by the mean (37-mer area
intensity/total integrated area intensity) of input DNA (the no protein lanes).
RESULTS
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Fig. 1.
Purification and characterization of the
I126D/L128E mutant of PCNA. A, purification to
homogeneity. Coomassie Blue-stained SDS-PAGE gel shows 4 µg of
purified human wild type (lane 1) or I126D/L128E mutant (lane 2) PCNA. The migration
positions of molecular mass markers (in kDa) are indicated.
B, the I126D/L128E mutant of PCNA does not bind to FEN-1.
Nickel-charged metal chelate resin was incubated with cleared bacterial
lysate from IPTG-induced cells containing a His6-tagged
human FEN-1 expression plasmid (lanes 1 and 2) or
vector alone as a control (lanes 3 and 4). An
equal aliquot of lysate from bacteria expressing wild type (lanes
1 and 3) or I126D/L128E mutant (lanes 2 and
4) PCNA was added to each assay. After washing, protein
complexes were denatured, separated by SDS-PAGE, and detected by
Coomassie Blue staining. The expression levels and solubilities of wild
type and I126D/L128E mutant PCNA were similar, as shown by SDS-PAGE
analysis of 7 µl of each induced cell lysate (lanes 5 and
6, respectively). C, the I126D/L128E mutant of
PCNA does not stimulate DNA polymerase activity. DNA polymerase
activity is shown as a function of wild type (filled
circles) or mutant (open circles) PCNA added per
reaction. Polymerase activity was quantitated as the incorporation of
32P-labeled TTP nucleotide into a poly(dA)/oligo(dT)
template/primer.
(void volume)/(solvent accessible volume) and which is
inversely proportional to log MW, was 0.389 for wild type PCNA and
0.384 for mutant PCNA. Bovine serum albumin, which at 66 kDa is smaller
than the 87-kDa PCNA trimer but larger than the 29-kDa PCNA monomer,
was used as a standard and gave a K value of 0.405.
(Fig.
1C). Compared with wild type PCNA, the activity of the mutant in this assay was negligible. This defect is the result of an
inability of the mutant PCNA to bind to DNA polymerase
. Recently,
very similar mutations have been characterized independently in yeast
and human PCNA (18, 25). In human PCNA, the L126S and I128A mutations
are moderately and severely impaired, respectively, in their abilities
to interact with DNA polymerase
(25). Likewise, the I126A/L128A
double mutant of yeast PCNA is severely defective in physical
interaction with DNA polymerase
, and must be used at concentrations
1000-fold greater than wild type PCNA to stimulate DNA polymerase
-mediated DNA synthesis (18). However, this mutant retains normal
functional interactions with RF-C, a 5-subunit complex that loads PCNA
onto the DNA primer-template junction in an ATP-dependent
manner. Interestingly, the I126A/L128A yeast PCNA mutant retains
binding to yeast FEN-1 (18), in contrast to the FEN-1-binding defect
reported here for the I126D/L128E human PCNA mutant. Residues 126 and
128 line a hydrophobic pocket of PCNA that interacts with protruding
hydrophobic residues of PCNA-binding ligands p21 (24) and, by analogy,
FEN-1 (16, 17). Thus, the introduction of charged acidic residues at
these positions more strongly interferes with the binding of
complementary hydrophobic FEN-1 residues at this site.
, and DNA ligase
activities. BER reaction intermediates were observed by selectively
omitting various components of the reconstituted system (Fig.
2). In the first step of BER, AP
endonuclease makes an incision immediately 5' of the abasic site,
producing a nick in the damaged strand. This nick leaves a 3'-hydroxyl
that serves as the primer for repair synthesis, and a 5'-terminal AP
site that must be excised before repair can be completed. In order to
permit observation of modifications to either side of the nick, 32P label was incorporated either 5 nucleotides to the 5'
side of the abasic site (5'-labeled; Fig. 2A) or 10 nucleotides to the 3' side of the abasic site (3'-labeled; Fig.
2B) within a covalently closed circular DNA substrate. After
termination of the repair reaction, HinfI restriction
digestion was used to release the 32P-labeled DNA for
analysis by gel electrophoresis. Thus, incision by AP endonuclease
cleaves the HinfI-HinfI DNA segment into two fragments that can be observed selectively in accordance with the
placement of the 32P label. AP endonuclease recognized and
incised the 3-hydroxy-2-hydroxymethyltetrahydrofuran lesion very
efficiently, as virtually all of the 32P-labeled substrate
was nicked to produce 5'-labeled (Fig. 2A, lane 1) or
3'-labeled (Fig. 2B, lane 1) incised fragment. The 3'-terminal hydroxyl of the 5'-32P-labeled incised
fragment was extended via repair synthesis in the presence of dNTPs,
BE-1 (providing RF-C and DNA polymerase
), and wild type PCNA (Fig.
2A, lane 3). As expected, mutant PCNA was unable to support
DNA synthesis (Fig. 2A, lane 4). Complete repair to yield
the ligated full-length HinfI-HinfI DNA fragment was very efficient with all of the constituents of the repair reaction
present (Fig. 2A, lane 6).
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Fig. 2.
Analysis of repair intermediates of wild type
or mutant PCNA. A, repair of the circular DNA substrate
labeled with 32P on the 5' side of the synthetic AP site.
B, repair of the circular DNA substrate labeled with
32P on the 3' side of the synthetic AP site. Repair
reactions were conducted as described under "Experimental
Procedures," with various combinations of proteins in the absence or
presence of dNTPs as indicated. Proteins used in these experiments were
AP endonuclease (AP endo.), FEN-1, protein fraction BE-1
(containing RF-C, DNA polymerase , and DNA ligase), and wild type
(wt) or I126D/L128E mutant (mut.) PCNA. After
termination of the repair reaction, DNA was digested with
HinfI, electrophoresed in an 8 M urea-containing
20% polyacrylamide gel, and subjected to autoradiography. Positions of
repaired products and intermediates are indicated for each
autoradiogram. The ligation intermediate is a product resulting from
addition of AMP to the 5'-terminal AP site of the incised fragment by
DNA ligase. Diagrams indicate the relative positions of 32P
label, the AP site, and the HinfI restriction sites for each
panel.
and FEN-1, produced reaction intermediates that indicated both a
repair synthesis defect (Fig. 2A) and an excision defect (Fig. 2B). This second observation suggested that the
excision step of BER may be facilitated by PCNA and, furthermore, that this activity of PCNA may be mediated through direct physical interaction with FEN-1. To further investigate this possibility, a
series of mutations was introduced into FEN-1 and the ability of the
mutant proteins to bind to PCNA was evaluated.2 From these
studies, the F343A/F344A double point mutant derivative of FEN-1 was
identified. By analogy with p21, the substituted phenylalanines were
predicted to be critical for the interaction of FEN-1 with the
hydrophobic ligand-binding pocket of PCNA.
= [FEN-1],
a = kobs(max), and
b = [FEN-1] at 1/2 kobs(max)) (20, 21). The best fit curve indicates a saturating
kobs of 0.07 s
1 for both the wild
type and mutant enzymes, in good agreement with previous estimates of
the cleavage rate constant for wild type FEN-1 (20). Half-maximal
binding was 173 nM (±45, S.E.) for wild type and 136 nM (± 51) for mutant FEN-1, showing that substrate
affinities were identical within the limits of experimental error. Wild
type and mutant FEN-1 were also compared using 32P-labeled
5'-flap DNA as substrate so that reaction products could be analyzed by
gel electrophoresis and autoradiography. These products were of the
same size and intensity, indicating no difference in cleavage site
preference between the two proteins (data not shown). In an exonuclease
assay using nicked substrate, the activities of wild type and
F343A/F344A mutant FEN-1 were again indistinguishable (Fig.
5). 5'-3' exonucleolytic degradation from
the nick site yielded an identical pattern of reaction products
regardless of protein used. Enzymatic specific activities were the same
as determined by 2-fold serial dilution of proteins over the
concentration range of interest. The amount of undegraded substrate
remaining after reaction with wild type FEN-1 at 1, 2, or 4 µM was 90.2, 71.3, and 11.8%, respectively, whereas
substrate remaining after reaction with mutant FEN-1 at 1, 2, or 4 µM was 95.5, 80.6, and 12.4%. Thus, the introduction of
the F343A/F344A mutation selectively modified FEN-1 such that binding
to PCNA was abolished while all aspects of catalytic activity were
unaffected. Because the catalytic and PCNA-binding regions of FEN-1 are
well defined and distinct (16, 17), it is not surprising that such
specificity can be achieved.
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Fig. 3.
Purification and characterization of the
F343A/F344A mutant of FEN-1. A, purification to near
homogeneity. Coomassie Blue-stained SDS-PAGE gel shows 7 µg of
purified His6-tagged human wild type (lane 1) or
F343A/F344A mutant (lane 2) FEN-1. The migration positions
of molecular mass markers (in kDa) are indicated. B, the
F343A/F344A mutant of FEN-1 does not bind to PCNA. Nickel-charged metal
chelate resin was incubated with cleared bacterial lysate from
IPTG-induced cells containing vector alone (lane 1), wild
type (lane 2), or F343A/F344A mutant (lane 3)
His6-tagged FEN-1 expression plasmid. An equal aliquot of
lysate from bacteria expressing wild type PCNA was added to each assay
(lanes 1-3). After washing, protein complexes were
denatured, separated by SDS-PAGE, and detected by Coomassie Blue
staining.
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Fig. 4.
The F343A/F344A mutant of FEN-1 has wild type
endonuclease activity. Beads bearing immobilized
5'-fluoresceinated flap DNA substrate were incubated with purified
enzyme at room temperature, and bead-associated fluorescence was
monitored as a function of time by flow cytometry. Assays were
conducted using wild type (A) or F343A/F344A mutant
(B) FEN-1 at 360 nM (filled circles),
180 nM (open circles), 36 nM
(filled triangles), 18 nM (open
triangles), 7.2 nM (filled squares), or 3.6 nM (open squares). Substrate fluorescence was
stable over time in the absence of enzyme (filled diamonds).
C, the observed first-order rate constant based on the time
to achieve 50% substrate consumption is shown for wild type
(filled circles) and F343A/F344A mutant (open
circles) FEN-1 at each enzyme concentration. A hyperbolic
substrate binding model (described under "Results") was used to
generate a best fit curve with equal weighting to each enzyme
concentration for wild type (solid line) and F343A/F344A
mutant (dashed line) FEN-1 rate constant data.
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Fig. 5.
The F343A/F344A mutant of FEN-1 has wild type
exonuclease activity. The exonuclease activity of wild type and
mutant FEN-1 was assayed using a linear double-stranded oligonucleotide
substrate bearing a single nick on one strand. As illustrated in the
diagram, a 32P label was incorporated at the 3'-end of the
strand that was susceptible to degradation by the 5'-3' exonuclease
activity of FEN-1. The DNA substrate was incubated with no protein
(lanes 1 and 5), wild type FEN-1 (lanes
2-4), or F343A/F344A mutant FEN-1 (lanes 6-8) at
enzyme concentrations of 1 µM (lanes 2 and
6), 2 µM (lanes 3 and
7), or 4 µM (lanes 4 and
8). Reaction products were electrophoresed in a 7 M urea-containing 15% polyacrylamide gel, and subjected to
autoradiography. The specific activities and exonuclease cleavage site
preferences of wild type and mutant FEN-1 were essentially
identical.
, and DNA ligase) plus varying
amounts of FEN-1. Repair synthesis was suppressed by omission of dNTPs
(Fig. 6, left panel) or permitted by dNTP inclusion (Fig. 6,
right panel). All reaction intermediates are the same as
those described for Fig. 2B. In the absence of FEN-1, two
types of pre-excision intermediates accumulate: the incised product and
the ligation intermediate (Fig. 6, lanes 1 and
12). As noted earlier (Fig. 2B), AP endonuclease cleaves the substrate at the AP site to generate the incised fragment, some of which undergoes 5'-phosphoryl-AMP modification by ligase to
form the ligation intermediate. These pre-excision reaction intermediates could be further processed by FEN-1 to give an excised fragment (Fig. 6, left panel) or repaired product (Fig. 6,
right panel). The excision step was
PCNA-dependent, as demonstrated earlier (compare
lanes 2 and 3 of Fig. 2B). Mutant
FEN-1 was markedly deficient at PCNA-dependent excision
(Fig. 6, left panel) and complete repair (Fig. 6,
right panel) over a range of concentrations up to 1 ng/reaction. The BER concentration dependence curve for mutant FEN-1
was shifted to the right by about 1 log unit compared with that of wild
type FEN-1. This indicates that the PCNA binding-defective mutant had
only one-tenth the specific activity of wild type FEN-1 in supporting
complete repair, even though basal catalytic activity of the two
proteins was identical (Figs. 4 and 5). These results show that the
physical interaction of PCNA and FEN-1 was required for efficient
excision and repair.
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Fig. 6.
Analysis of repair intermediates of wild type
or mutant FEN-1 in the PCNA-dependent pathway. All
reactions used the circular DNA substrate labeled with 32P
on the 3' side of the synthetic AP site, as illustrated in Fig.
2B. Repair reactions were conducted as described under
"Experimental Procedures," in the absence (lanes 1-11)
or presence (lanes 12-22) of dNTPs, with AP endonuclease,
wild type PCNA, and protein fraction BE-1 (containing RF-C, DNA
polymerase , and DNA ligase) present in each. Reactions included
either wild type (wt; lanes 2-6 and 13-17) or
F343A/F344A mutant (mut; lanes 7-11 and 18-22)
FEN-1 as follows: no FEN-1 (lanes 1 and 12), 0.01 ng (lanes 2, 7, 13, and 18), 0.03 ng (lanes
3, 8, 14, and 19), 0.1 ng (lanes 4, 9, 15, and 20), 0.3 ng (lanes 5, 10, 16, and
21), and 1 ng of FEN-1 (lanes 6, 11, 17, and
22). After termination of the repair reaction, DNA was
digested with HinfI, electrophoresed in an 8 M
urea-containing 20% polyacrylamide gel, and subjected to
autoradiography. Positions of repaired products and intermediates are
indicated on the side of the autoradiogram. The excised product
(left panel) and repaired product (right panel)
were quantitated with a phosphorimage analyzer and plotted as percent
of total radioactivity for wild type FEN-1 (filled circles)
and mutant FEN-1 (open circles).
-Dependent Repair System--
Recently, it has been
shown that AP endonuclease, DNA polymerase
, and DNA ligase can
process reduced AP site analogs if the reaction is supplemented with
FEN-1 (6, 7). Using X. laevis proteins, we have demonstrated
that FEN-1 can excise a 5'-terminal AP site from the flap structure
formed by DNA synthesis with DNA polymerase
, and that this excision
reaction does not require PCNA (7). We tested whether F343A/F344A
mutant FEN-1 still retained this PCNA-independent excision activity. At
10 ng/reaction, neither wild type nor mutant FEN-1 exhibited excision activity in the absence of dNTPs (Fig.
7A, lanes 1-3). This
contrasts with the excision activity observed at much lower
concentration (0.1-1 ng/reaction) for wild type but not mutant FEN-1
under similar conditions in the PCNA-dependent system (Fig.
6, left panel). When dNTPs were included to permit
strand-displacing synthesis by DNA polymerase
(Fig. 7B),
both wild type and mutant FEN-1 proteins efficiently removed
approximately 5 nucleotides that included the AP site. The number of
nucleotides excised by FEN-1 was in a good agreement with the length of
DNA synthesis catalyzed by DNA polymerase
in a processive manner
(26, 27).
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Fig. 7.
Analysis of repair intermediates of wild type
or mutant FEN-1 in the DNA polymerase
-dependent pathway. All reactions
used the circular DNA substrate labeled with 32P on the 3'
side of the synthetic AP site, as illustrated in Fig. 2B.
Positions of repaired products and intermediates are indicated for each
autoradiogram. A, effect of DNA synthesis by DNA polymerase
on FEN-1 activity. Repair reactions were conducted as described
under "Experimental Procedures," with various combinations of
proteins in the absence or presence of dNTPs as indicated. Proteins
used in these experiments were AP endonuclease (AP endo.),
DNA polymerase
(Pol
), and wild type (wt)
or F343A/F344A mutant (mut) FEN-1. After termination of the
repair reaction, DNA was digested with HinfI,
electrophoresed in an 8 M urea-containing 20%
polyacrylamide gel, and subjected to autoradiography. B,
titration of FEN-1 proteins. Reactions were conducted in the presence
of dNTPs with AP endonuclease, DNA polymerase
, and either wild type
(wt; lanes 3-7 and 14-18) or F343A/F344A mutant
(mut; lanes 8-12 and 19-23) FEN-1 as follows:
no FEN-1 (lanes 2 and 13), 0.01 ng (lanes
3, 8, 14, and 19), 0.03 ng (lanes 4, 9, 15, and 20), 0.1 ng (lanes 5, 10, 16, and
21), 0.3 ng (lanes 6, 11, 17, and 22),
and 1 ng of FEN-1 (lanes 7, 12, 18, and 23). In
lanes 13-23, 10 ng of wild type PCNA was added to the
reactions. In lane 1, the DNA substrate was treated with AP
endonuclease only to show the incised product. Reaction products were
analyzed as described for panel A. Excised product was
quantitated with a phosphorimage analyzer and plotted as percent of
total radioactivity for wild type FEN-1 (filled circles) and
mutant FEN-1 (open circles).
DISCUSSION
and
.
The involvement of PCNA in DNA damage repair has been documented for
nucleotide excision repair (30, 31), mismatch repair (32-34),
double-strand break repair (35), and long-patch BER (7-9, 11, 12).
Repair synthesis mediated by PCNA, RF-C, and DNA polymerase
or
has been shown for many of these processes. Recently, additional
functions for PCNA in repair have begun to emerge. PCNA acts in repair
synthesis and also at an early presynthesis step in mismatch repair
(32-34), and its interaction with the repair endonuclease XPG has
functional significance during nucleotide excision repair (17). Here we
show that the FEN-1 binding activity of PCNA is important in BER and,
furthermore, that this interaction serves to facilitate excision.
-catalyzed elimination reaction removes the 5'-terminal AP site
only, whereas FEN-1-catalyzed phosphodiester bond hydrolysis releases
two or more nucleotides to yield the patch size that defines long-patch
BER. The best substrate for FEN-1 nuclease activity in vitro
is a 5'-flap structure (39, 40), which has a displaced single strand
bearing a free 5'-end and an annealed upstream primer abutting the
single-strand/double-strand transition. Strand-displacing synthesis
during BER could create this favored 5'-flap substrate, thereby
allowing excision by FEN-1 endonuclease action. Such an endo mechanism
is clearly involved in the DNA polymerase
-dependent BER
system, because excision was strictly dependent upon strand-displacing
synthesis (Fig. 7A).
-dependent assay, wild type and mutant FEN-1 at 0.1-1
ng/reaction were equally efficient at excising the displaced strand
(Fig. 7B). In the PCNA-dependent BER pathway, it
appears that wild type FEN-1 makes use of a more efficient exo
mechanism predominantly, whereas mutant FEN-1 must use a less efficient
endo mechanism. If this explanation is correct, BER exonuclease
activity requires interaction with PCNA.
alone or the PCNA-DNA polymerase
complex are capable of
providing repair synthesis activities for long-patch BER (6, 7). With
respect to excision, these two systems appear to be mechanistically
distinct, as the former requires strand-displacing DNA synthesis
whereas the latter does not. In long-patch BER assay systems that use
cell extracts, PCNA dependence and DNA polymerase
-independence are
both evident (9, 11, 12). Such studies confirm the physiological
relevance of the PCNA-dependent pathway for long-patch BER.
The new result that PCNA functionally interacts with the essential
long-patch repair protein FEN-1 further supports this view. In
addition, we have found that yeast strains possessing a mutant
rad27 allele that is specifically defective in binding to
PCNA grow slowly in the presence of methyl methanesulfonate, suggesting
a role for the PCNA-FEN-1 interaction during BER in vivo.3
(41), providing a recruitment mechanism for the next enzyme in the sequence of events. After provision of dRP elimination and single nucleotide repair synthesis by DNA polymerase
, ligation by either DNA ligase I
or the DNA ligase III/XRCC1 heterodimer completes repair. The DNA
ligase III/XRCC1 heterodimer appears to be the relevant short-patch BER
ligase in cell extracts (42), and it binds directly to DNA polymerase
(43). Thus, a complete chain of physical interactions is known
among short-patch BER proteins that are necessary and sufficient to
process the AP site completely. These interactions no doubt increase
the efficiency of repair by placing repair enzymes at the appropriate
sites in the appropriate sequence, thereby coordinating repair events.
An important consequence of such coordination may be that repair
intermediates exist only transiently in vivo. Nucleotide
gaps and unligated nicks, if allowed to persist while awaiting further
processing, might be subject to nonspecific exonuclease action, and
would likely be recombinogenic DNA structures as well. The
high-efficiency BER processing aided by protein-protein interaction may
be important in suppressing such unwanted secondary events. A similar
situation may exist in long-patch BER as well. A chain of
protein-protein interactions that facilitates long-patch BER is
beginning to unfold, with PCNA as the central element. PCNA binds to
and functionally interacts with RF-C and DNA polymerase
, and these
proteins together can perform long-patch BER repair synthesis (6-8).
PCNA also binds to the essential long-patch BER nuclease FEN-1, an
interaction that is functionally important, as shown here. Ligation
during long-patch BER probably utilizes DNA ligase I, because XRCC1
mutant cell extracts exhibit a ligation defect affecting DNA polymerase
-dependent but not PCNA-dependent BER (42),
indicating that the DNA ligase III/XRCC1 heterodimer does not act in
the latter pathway. PCNA binds to DNA ligase I (44, 45), so this ligase
possesses a potential recruitment mechanism that would continue the
protein-protein interaction chain that centers about PCNA during
long-patch BER.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. John Nolan for FEN-1 enzymatic
assay data analysis, and Dr. Suman Lee for characterization of the
FEN-1 cleavage site in the 5'-flap endonuclease assay. We also thank
Drs. Chen-Keat Tan and Antero G. So for providing calf thymus DNA
polymerase .
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FOOTNOTES |
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* This work was supported by the Office of Biological and Environmental Research of the U. S. Department of Energy and National Institutes of Health Grants CA71630 (to M. S. P.) and CA63154 (to Y. M.).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.
§ To whom correspondence should be addressed: Life Sciences Div., M888, Los Alamos National Laboratory, Los Alamos, NM 87545. Tel.: 505-665-7015; Fax: 505-665-3024; E-mail: gary{at}telomere.lanl.gov.
The abbreviations used are:
BER, base
excision repair; AP, apurinic/apyrimidinic; dRP, deoxyribose phosphate; FEN-1, flap endonuclease-1; PCNA, proliferating cell nuclear antigen; RF-C, replication factor C; PAGE, polyacrylamide gel electrophoresis; PAM, percentage of accepted point mutations; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol; IPTG, isopropyl-1-thio--D-galactopyranoside.
2 R. Gary and M. Park, unpublished data.
3 R. Gary and D. Gordenin, unpublished data.
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REFERENCES |
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