Proc. Natl. Acad. Sci. USA
Vol. 94, pp. 7166–7169, July 1997
Interaction of human apurinic endonuclease and DNA polymerase
? in the base excision repair pathway
RICHARD A. O. BENNETT*, DAVID M. WILSON III*†, DONNY WONG, AND BRUCE DEMPLE‡
Department of Molecular and Cellular Toxicology, Harvard School of Public Health, Boston, MA 02115
Communicated by Charles C. Richardson, Harvard Medical School, Boston, MA, May 5, 1997 (received for review April 11, 1997)
directly by DNA-damaging agents or by DNA glycosylases
acting in base excision repair. AP sites are corrected via
incision by AP endonucleases, removal of deoxyribose 5-phos-
phate, repair synthesis, and ligation. Mammalian DNA poly-
merase ? (Pol?) carries out most base excision repair syn-
thesis and also can excise deoxyribose 5-phosphate after AP
endonuclease incision. Yeast two-hybrid analysis now indi-
cates protein–protein contact between Pol? and human AP
endonuclease (Ape protein). In vitro, binding of Ape protein to
and the AP-DNA. After incision by Ape, only Pol? exhibits
stable DNA binding. Kinetic experiments indicated that Ape
accelerates the excision of 5?-terminal deoxyribose 5-phos-
phate by Pol?. Thus, the two central players of the base
excision repair pathway are coordinated in sequential reac-
Mutagenic abasic (AP) sites are generated
DNA is subject to spontaneous hydrolytic degradation and
assault by metabolic byproducts, such as oxygen radicals and
alkylating agents, as well as numerous environmental agents
(1, 2). Genetic instability can result from this damage and is
counteracted by various pathways of DNA repair. Nucleotide
excision repair acts on a wide variety of DNA lesions and is
mediated in mammalian cells by some 25 proteins acting in an
orchestrated fashion (3, 4). Base excision repair (5) handles a
more limited set of altered bases through the action of DNA
(6, 7). DNA glycosylases produce AP sites, which also arise
from spontaneous loss of normal bases, destabilization by base
damage, or base elimination by free radical attack (1, 2).
Abasic (AP) sites threaten genetic stability because they block
replication and are mutagenic (8).
Both direct and glycosylase-generated AP sites are sub-
strates for AP endonucleases, which initiate repair by incising
immediately 5? to the AP site (5). The major AP endonuclease
of human cells, Ape protein (also called Ref1, Hap1, or Apex),
is a member of a large family of nucleases related to exonu-
clease III of Escherichia coli (5, 9). Ape protein has broad
specificity for AP sites (10) and is abundantly present in the
nucleus of human cells (11, 12). The next step of base excision
repair is the removal of 5?-terminal deoxyribose 5-phosphate
(dRp), the product of Ape, followed by DNA repair synthesis
to fill in the small gap, and ligation to complete the repair (1).
In mammalian cells, most of the repair synthesis is carried out
by DNA polymerase ? (Pol?) (13, 14). A distinct dRp excision
activity has been recovered in small amounts from calf thymus
(15). However, a recent report demonstrated that Xenopus
DNA Pol? has an intrinsic activity that releases 5?-dRp by
?-elimination (16); this mechanism has been confirmed for
human Pol? (17). This finding suggested that one protein can
catalyze consecutive steps (dRp removal and repair synthesis)
in the base excision pathway. Both the nucleotide excision (3,
4) and DNA mismatch repair (1) pathways act by coordinating
protein activities. We wished to determine whether base
excision repair also might proceed in an orchestrated fashion
mediated by specific protein–protein interactions.
MATERIALS AND METHODS
Two-Hybrid Methods. The APE cDNA (12) was amplified
by PCR (18) and inserted in-frame into the vector pAS1 (19)
to generate pAS1–Ape, which encodes a Gal4DB–Ape fusion
protein. Similarly, the Pol? cDNA (14) was amplified and
inserted in-frame in the vector pACT2 to generate pACT-
Pol?, encoding a Gal4AD–Pol? fusion protein. The structures
of the fusion constructs were confirmed by DNA sequencing.
Plasmid pAS1–Ape was transformed into yeast strain Y190
(MATa gal4 gal80 his3 trp1–901 ade2–101 ura3–52 leu2, 3–112
pACT-Pol? or pSE1111 then were introduced and the cells
plated on synthetic medium lacking tryptophan, leucine, and
histidine, and containing 25 mM 3-amino-1,2,4-triazole (19).
After incubation at 30°C for 5 days, colonies were transferred
to a nitrocellulose membrane by blotting and stained for
?-galactosidase activity (19). For cell extracts to assay ?-ga-
lactosidase activity, 20-ml cultures were grown ?3 days in
selective medium as above. The cells were collected, lysed by
shaking with glass beads (500 ?m diameter), and the extracts
assayed using the fluorogenic substrate 4-methylumbelliferyl
?-D-galactoside (21). The positive control was a yeast strain
carrying the E. coli lacZ gene on a plasmid (R. Brennan and
R. H. Schiestl, personal communication).
Electrophoretic Mobility-Shift Assay (EMSA). Oligonucle-
otides containing either a uracil or a tetrahydrofuran residue
(23-F) were32P-5?-end labeled and annealed to unlabeled
complementary DNA as previously described (22). AP site
DNA (18-AP and 51-AP) was generated by incubating uracil-
containing substrates with 1 unit of uracil DNA glycosylase
(GIBCO?BRL) for 5 min at 37°C. For a typical binding
(where indicated) with Ape protein purified from HeLa cells
(11) (20 ng, ?55 nM) or human Pol? (23) (20 ng, ?55 nM) in
50 mM Hepes?KOH, pH 7.5, 100 mM KCl, 10% glycerol, and
either 10 mM MgCl2 (Mg) or 4 mM EDTA (EDTA). The
binding reaction (final volume 10 ?l) was incubated at 0°C for
5 min and resolved on a 8% nondenaturing polyacrylamide gel
(22) followed by autoradiography. In ‘‘supershift’’ experi-
ments, the proteins and DNA were incubated as described
above, and the appropriate rabbit polyclonal antiserum, gen-
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© 1997 by The National Academy of Sciences 0027-8424?97?947166-4$2.00?0
PNAS is available online at http:??www.pnas.org.
Abbreviations: AP, abasic; Pol?, DNA polymerase ?; Ape, AP
endonuclease; dRp, deoxyribose 5-phosphate; EMSA, electrophoretic
*R.A.O.B. and D.M.W. contributed equally to this work.
†Present address: Biology and Biotechnology Research Program,
Lawrence Livermore National Laboratory, Livermore, CA 94551.
‡To whom reprint requests should be addressed. e-mail: demple@
erated against purified Ape from HeLa cells (11, 12) or
recombinant human Pol? (S. Linn, personal communication),
was then added, and the incubation continued for 20 min at
0°C. The mixtures then were resolved on nondenaturing gels as
dRp Substrate and Excision Assay. An oligonucleotide
containing a dUMP residue at position 22 was 3?-labeled with
dideoxy-[?-32P]ATP using terminal deoxynucleotidyltrans-
ferase and annealed to the complementary oligonucleotide
(13). For each experiment, the double-stranded substrate
(typically 10 pmol in 30 ?l) was treated at 37°C for 30 min with
60 units of purified E. coli uracil-DNA glycosylase (kindly
provided by D. Mousbaugh, Oregon State University) to
create an AP site at position 22 in the uracil-containing strand
(13). For each dRp excision reaction, 0.5 pmol of the newly
formed AP site-containing substrate was cleaved to comple-
tion (?95%) by incubation for 10 min at 37°C in an 8-?l
reaction with 6 ng of purified endonuclease IV (24) in 50 mM
Hepes?KOH, pH 7.5?50 mM NaCl?2 mM DTT?2.5% glycer-
ol?100 ?g/ml BSA. Purified Pol? (9 fmol) and 0, 8, 80, or 800
fmol of Ape protein were added, and the mixtures (final
volume 10 ?l) were incubated at 22°C. At the indicated times,
samples were removed from the reactions and treated with
NaBH4(final concentration 340 mM) to stabilize the AP sites
(16). The samples were ethanol-precipitated, resuspended in
H2O, mixed with an equal volume of formamide-containing
loading buffer, and analyzed on a denaturating 20% polyacryl-
amide gel (18).
We initially tested for interaction between Ape and Pol? using
the yeast two-hybrid system (25). Fusions of Ape to the yeast
Gal4 DNA-binding domain (Gal4DB–Ape) and of Pol? to the
Gal4 transactivation domain (Gal4AD–Pol?) were coexpressed
in Saccharomyces cerevisiae bearing appropriate Gal4-
genes by reconstitution of Gal4 activity was observed in cells
coexpressing the Gal4DB–Ape and the Gal4AD–Pol? fusions
(Fig. 1). No reporter gene activation was observed when the
Gal4DB–Ape fusion was expressed alone or with a different
protein fused to the Gal4 transactivation domain (Gal4AD–
Snf4 encoded by pSE1111, ref. 19; Fig. 1). Direct assay of
?-galactosidase showed strong activation of the lacZ reporter
gene in six independent transformants coexpressing GalDB–
Ape and Gal4AD–Pol? (Fig. 1). Two-hybrid analysis gave no
indication that Ape interacts with another component of the
base excision repair pathway, human alkylpurine-DNA glyco-
sylase (26). Additional control experiments found no evidence
for nonspecific interactions of Pol? with other proteins (yeast
Snf1 (19) or Epstein–Barr virus LMP1 (27) (data not shown).
Thus, Ape and Pol? interact specifically in vivo.
We next sought in vitro evidence for the interaction of Ape
and Pol?. Because these two proteins act in consecutive steps
of base excision repair, we tested whether they might interact
on a DNA substrate. We recently have shown that Ape protein
can form stable complexes with uncleaved AP sites in DNA
(22). Such complexes are detectable by filter binding or EMSA
and are enhanced in the presence of the metal chelator EDTA
(22). In the experiments shown here, Ape?AP–DNA com-
plexes were seen by EMSA for 18-bp, 23-bp, and 51-bp duplex
oligonucleotides containing AP sites (Fig. 2A, upper bands;
lower bands correspond to the free DNA). The subsequent
addition of purified Pol? produced complexes of slightly
slower mobility in EMSA than those found with Ape alone
(Fig. 2A). When Mg2?was added to support Ape incision
activity (22), no complex of Ape alone or with Pol? was
detected with the 18-bp substrate (Fig. 2A), probably due to
denaturation of the short (9- and 8-nucleotide) strands after
cleavage. However, incubations with the 51-mer generated a
substantial amount of complex in Mg2?when both Ape and
Pol? were present. This result shows that, as expected, Ape
alone does not remain bound to an incised AP site, although
Pol? can bind such a site strongly. This experiment did not
resolve whether Ape remained bound after cleavage in the
presence of Pol?, or whether both proteins are present in the
complexes formed under noncleavage conditions.
These questions were addressed using Ape-specific and
Pol?-specific antisera in EMSAs. In EDTA, Ape and Pol?
together formed complexes (middle bands in Fig. 2B) that
were super-shifted quantitatively when antibody against either
protein was included in the incubation. Antibody binding
formed complexes that were substantially more retarded in the
gel than those formed by Ape and Pol? alone (upper ‘‘super-
shifted’’ bands in Fig. 2B). We noted that a greater proportion
of the AP substrate was bound in protein-DNA complexes in
the presence of antibodies. Perhaps antibody binding stabilizes
the complexes of Ape and Pol? with the DNA. A minor
fraction of the Ape–DNA complex was further shifted by the
anti-Pol? antiserum (Fig. 2B, Left), which indicates a weak
crossreactivity. In the presence of Mg2?, super-shifting was
observed only with the anti-Pol? antibodies, even though both
Pol? and Ape were present in the binding reaction (Fig. 2B);
the anti-Ape antibodies evidently do not crossreact with Pol?
pAS1–Ape encodes the Gal4DB–Ape fusion; pACT–Pol? encodes the Gal4AD–Pol? fusion; pSE1111 encodes a Gal4AD-Snf4 fusion; pSE1112
encodes a Gal4DB-Snf1 fusion (19). (Center) Indicator plate for ?-galactosidase expression from a GAL promoter-lacZ fusion and His?selection
from a GAL promoter-HIS3 fusion. (Right) Quantitation of ?-galactosidase expression by a fluorescent assay. Ape-DBD is the Gal4DB–Ape fusion,
Pol?-AD the Gal4AD–Pol? fusion. Six independent cotransformants of pAS1-Ape and pACT-Pol? were assayed for ?-galactosidase activity; single
isolates of the other transformants were assayed. An S. cerevisiae lacZ?strain (obtained from R. Brennan and R. H. Schiestl, Harvard School of
Public Health, Boston) was assayed as a positive control.
Interaction of Ape and Pol? in the yeast two-hybrid system. (Left) Schematic of plasmids present in indicator strain Y190 (20). Plasmid
Biochemistry: Bennett et al.Proc. Natl. Acad. Sci. USA 94 (1997)7167
in this assay. Thus, only Pol? remains stably bound to the
nicked DNA substrate in complexes resolved by EMSA. This
result does not rule out a looser association of Ape with Pol?
on the incised DNA.
The experiments of Fig. 2 indicated that Ape acts as a
loading factor for Pol? onto nonincised AP sites in DNA. The
next question was whether this interaction affects the enzy-
matic activities of either protein. Pol? at various concentra-
tions had no detectable effect on Ape’s AP endonuclease
activity (using various substrates; refs. 10 and 11) or 3?-repair
diesterase activity (11) (data not shown). However, Ape
stimulated 5?-dRp excision by Pol?. The release of dRp was
monitored using a 51-mer DNA substrate containing a central
AP site (13) previously incised with a catalytic amount of E.
coli endonuclease IV, which cleaves in the same position as
Ape (5). As shown previously (16), high-resolution gel elec-
trophoresis separates the 3?-labeled fragment bearing a 5?-dRp
moiety (Fig. 3A, upper bands) from the product without dRp
(Fig. 3A, lower bands). Using such a substrate, 9 fmol of Pol?
alone removed only 10% of the 5?-dRp during a 25-min
incubation. Addition of 8, 80, or 800 fmol of Ape protein
increased the rate of dRp excision by Pol? by 2.7-, 3.3-, and
4.3-fold, respectively, for the 25-min reaction (Fig. 3B). The
stimulating effect of Ape on the dRp-excision activity of Pol?
also was observed for 10-min reactions (Fig. 3A) and in several
independent experiments. The highest level of Ape protein
alone (800 fmol) exhibited little (?10% excision) or no
detectable dRp-releasing activity over many experiments (Fig.
of E. coli exonuclease III, the bacterial homolog of Ape (5), did
not stimulate dRp excision by Pol? (data not shown).
These experiments demonstrate a previously unrecognized
level of coordination in the mammalian base excision repair
pathway. A specific interaction of Ape and Pol? occurs that
assembles the polymerase onto an AP site in DNA. After AP
(0.55 pmol) or Pol? (0.55 pmol) were incubated with the indicated 5?-labeled AP substrates in reactions containing either 4 mM EDTA or 10 mM
MgCl2 (Mg2?), and the uncomplexed DNA (lowest bands) and protein DNA complexes (upper bands) resolved by electrophoresis in a
nondenaturing gel (22). Binding substrates were 23-F, a 23-bp duplex DNA containing a tetrahydrofuran residue (10); 18-AP, an 18-bp
oligonucleotide containing an AP site generated by uracil excision (10); and 51-AP, a 51-bp duplex oligonucleotide containing an AP site generated
by uracil excision (13). (B) Presence of Ape and Pol? in complexes. Binding reactions with EDTA or Mg2?were carried out with Ape or Pol?
and the 51-AP substrate as described above, then Ape-specific (?Ape) or Pol?-specific (??-pol) antisera were added. The complexes containing
Ape or Pol? (middle bands) were resolved from the antibody-supershifted complexes (top bands) by electrophoresis in nondenaturing gels. In no
case was significant material retained in the wells of the gels.
Loading of Pol? onto AP sites by Ape protein. (A) Binding to AP sites in different sequence contexts. Purified human Ape protein
a catalytic amount of E. coli endonuclease IV, then incubated for the indicated times with purified human Pol? (9 fmol) and varying amounts of
purified human Ape protein (0, 8, 80, or 800 fmol). After the incubation, the substrate bearing 5?-dRp (upper band) and the product after dRp
excision (lower band) were resolved in a denaturing gel. OH?, substrate hydrolyzed with NaOH; X, the 5?-dRp substrate before incubation; 15,
5?-dRp substrate incubated 15 min with 800 fmol of Ape alone. (B) Quantitation of dRp excision. The gel in A was subjected to scanning
densitometry and the ratio of the substrate (upper band in A) to product (lower band in A) used to calculate the excision of dRp in 25-min reactions.
Activation of Pol? dRp excision activity by Ape. (A) A duplex oligonucleotide containing an AP site at position 22 was cleaved with
7168 Biochemistry: Bennett et al. Proc. Natl. Acad. Sci. USA 94 (1997)
site cleavage by the endonuclease, the Ape-dependent stimu-
lation of Pol?’s dRp-excision activity then can accelerate the
next step in the pathway, and perhaps the subsequent repair
synthesis as well. In vivo, the relative amounts of Ape protein
molecules per cell) (28) are similar to the range used in our in
vitro experiments. An excess of Ape would favor productive
interaction with Pol?, such that AP sites would most likely
already be associated with a molecule of Ape before the
binding of Pol?.
A previous effort failed to identify the Ape–Pol? interaction
using affinity chromatography (29), and we also have been
unsuccessful in that approach. These negative results might
result from a requirement for the Ape–Pol? interaction to
occur in the context of DNA, as was the case for the EMSA
approach used here. In the two-hybrid experiments, the addi-
tional proteins of Gal4 and the transcriptional apparatus also
could act as a scaffold for the interaction (25).
There appear to be two branches of base excision repair in
mammalian cells that differ in the DNA polymerase required
(13, 30): a Pol?-dependent branch that repairs ?90% of the
AP sites and a DNA polymerase ?- or ?-dependent branch.
Recent observations indicate that Pol? also interacts with
DNA ligase III via XRCC1 protein (31, 32) and perhaps with
DNA ligase I (29). Interaction of Pol? with DNase V, a
bidirectional exonuclease, has been known for some time (33).
Thus, Pol? may participate in multiple protein–protein inter-
actions to coordinate steps of the base excision pathway.
Engineered ‘‘knock-out’’ of either Ape or Pol? generates an
embryonic-lethal phenotype without apparent tissue-specific
defects (14, 34). These observations indicate a crucial role for
base excision repair in animal viability. It is not known whether
viable Ape-deficient cell lines can be obtained. However,
Pol?-deficient mouse cells do grow in culture, where they
exhibit hypersensitivity to alkylating agents (14) consistent
with a critical role for Pol? in base excision repair. The
possibility also should be considered that some repair defects
might act by disrupting the coordinating interactions in base
excision repair without altering the intrinsic activities of Pol?
and Ape. Thus, the molecular basis of the Pol?–Ape interac-
tion needs to be defined in detail.
We are indebted to Dr. Steve Elledge for supplying the two-hybrid
system vectors and for much helpful advice, to Dr. Stuart Linn for
providing purified Pol? and Pol?-specific antiserum, to Dr. Dale
Mosbaugh for uracil-DNA glycosylase, and to Drs. R. Brennan and
R. H. Schiestl for the yeast strain expressing E. coli ?-galactosidase.
We are grateful to Drs. James C. Wang and Graham C. Walker for
helpful comments on the manuscript. The able assistance of John
Davidson in constructing pAS1-Ape is gratefully acknowledged. This
work was supported by an National Institutes of Health grant
(GM40000) to B.D., a pilot project grant to R.A.O.B. and D.M.W.
from the Kresge Center for Environmental Health, Harvard School of
Public Health, a National Research Service Award fellowship
(CA62845) to D.M.W., and National Institutes of Health training
grants supporting R.A.O.B. (CA09078 and ES07155) and D.W.
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