From the
The major apurinic/apyrimidinic (AP) endonuclease of human
cells, the Ape protein, incises DNA adjacent to abasic sites to
initiate DNA repair and counteract the cytotoxic and mutagenic effects
of AP sites. Here we address the determinants of Ape AP endonuclease
activity using duplex DNA substrates that contain synthetic analogs of
AP sites: tetrahydrofuranyl (F), propanediol (P), ethanediol (E), or
2-(aminobutyl)-1,3-propanediol (Q). The last of these, a branched
abasic structure, was a poor substrate for which Ape had k
Apurinic/apyrimidinic (AP)
A
characteristic form of oxidative DNA damage is free radical-initiated
breaks bearing 3`-phosphates or 3`-phosphoglycolate esters (Giloni et al., 1981; Henner et al., 1983; Demple et
al., 1986). These oxidative damages, along with the 3`-deoxyribose
products of DNA glycosylase-associated ``class I'' AP
endonuclease (
Two families of
``class II'' AP endonuclease/3`-repair diesterase enzymes
have been identified. Endonuclease IV of Escherichia coli and
Apn1 protein of the yeast Saccharomyces cerevisiae are
homologous nucleases (Popoff et al., 1990) that perform
demonstrable roles in correcting oxidative or alkylation damages in
vivo (Cunningham et al., 1986; Ramotar et al.,
1991). Yeast lacking Apn1 display a substantially increased spontaneous
mutation rate (Ramotar et al., 1991; Kunz et al.,
1994), which underscores the role of AP endonucleases in preventing
mutagenesis due to DNA damage caused by by-products of normal
metabolism.
The second family of AP endonuclease/3`-repair
diesterases includes E. coli exonuclease III and the
predominant human AP endonuclease called Ape protein ((Demple et
al., 1991); also called Hap1 (Robson and Hickson, 1991), Apex
(Seki et al., 1992), or Ref1 (Xanthoudakis et al.,
1992)). Exonuclease III has a clear in vivo repair function in E. coli (Demple et al., 1986; Cunningham et
al., 1986). Expression of the human AP endonuclease in E. coli can substitute for exonuclease III in repair of alkylation-induced
AP sites, but evidently not efficiently for repair of oxidative damage
(Demple et al., 1991; Robson and Hickson, 1991; Seki et
al., 1992). This pattern is consistent with the in vitro properties of Ape, which has a relatively low 3`-repair activity
(present at
Intact
Ape protein was prepared as follows. Overnight cultures of DW5281
(
For purification of glutathione S-transferase-Ape fusion protein, BL21 cells containing
pGEX-Ape construct were induced with
isopropyl-1-thio-
Enzymatic reactions utilizing
endonuclease IV (AP endonuclease activity of 80,000 units/mg; Levin et al.(1988)) or exonuclease III (22,000 units/mg; Life
Technologies, Inc.) with MgCl
The possibility that
the exonuclease detected in our experiments might be due to a
contaminant was addressed in immunological studies. A polyclonal rabbit
antiserum generated against highly purified Ape from HeLa cells (Chen et al., 1991) immunoprecipitated the exonuclease activity
associated with both HeLa-derived Ape protein and recombinant Ape
isolated from E. coli (Fig. 4A). It is very
unlikely that cross-reacting material other than Ape protein would be
present in both of these preparations. Thus, the weak 3`-exonuclease is
probably an activity of Ape itself.
A
mismatch placed immediately 5` to an F moiety reduced the rate of Ape
cleavage by
Activity of Ape
was also analyzed using a series of 18-bp duplex substrates with F
placed at 1-17 bases from the 5` terminus. Incision by Ape was
detected only when F was flanked by at least 4 base pairs 5` and 3 base
pairs 3` to the lesion (Fig. 6). Cleavage by Ape on the substrate
with F at position 15 (with 3 base pairs on the 3`-side) was
significantly diminished compared with the substrate with F at position
14 (Fig. 6). Analysis of these reactions using a DNA sequencing
gel (Sambrook et al., 1989) confirmed that little, if any,
cleavage was occurring with F at positions 1-4 or 16 and 17 (data
not shown).
The mode of substrate recognition by AP endonucleases has
been the subject of discussion for nearly 2 decades, initially in an
effort to account for the apparently diverse activities of bacterial
enzymes such as E. coli exonuclease III (Weiss, 1976). Despite
their limited exonuclease activity against undamaged DNA, the
eukaryotic class II AP endonucleases hydrolyze a broad array of
substrates ranging from intact AP sites to 3`-terminal nucleotide
fragments to 3`-phosphates (Demple and Harrison, 1994). The recognition
mechanism(s) of this class of enzymes must, therefore, accommodate
different types of DNA damage, seemingly in all sequence contexts,
while ignoring the structural fluctuations of normal DNA. The studies
presented here represent the first systematic effort to define key
elements of substrate recognition and incision by Ape, the main AP
endonuclease of human cells.
The substrate profile for Ape was
similar to that found for E. coli exonuclease III in the
presence of Ca
A detailed assessment of the
structural requirements for Ape acting on abasic sites is warranted,
since different types of abasic sites may be formed under different
circumstances. In addition to AP sites formed by acid- or
glycosylase-catalyzed base hydrolysis (Lindahl and Nyberg, 1974;
Wallace, 1988; Demple and Harrison, 1994), free radical agents such as
x-rays (von Sonntag, 1987) or bleomycin (Steighner and Povirk, 1990)
form modified abasic sites containing oxidized forms of deoxyribose.
Enzymes such as the Ape protein may have to cope with these lesions as
the products of metabolic and environmental mutagens. Indeed, recent
work has shown that exonuclease III and endonuclease IV of E.
coli, which represent the two families of ``class II''
AP endonucleases (Demple and Harrison 1994), distinguish strongly
between abasic sites oxidized at the 1 or the 4 position (Häring et al., 1994). It remains to be determined how Ape protein
acts on these modified sites.
The duplex DNA context is of
importance for Ape, as it is for exonuclease III (Takeuchi et
al., 1994). As suggested by Weiss(1976), the double-helical
structure on the 5`-side of the abasic site is much more important than
that on the 3`-side. Thus, Ape protein requires at least four base
pairs 5` to the lesion, but no more than three base pairs on the
3`-side. Ape and exonuclease III may be able to cleave duplex
substrates as short as eight base pairs, although neither enzyme
attacks abasic sites in single-stranded DNA.
Consistent with the
evident requirement that AP endonucleases act on damages produced
anywhere in the genome, the nature of the nucleotide opposite the
abasic site is essentially irrelevant. An exception was the case where
F was opposed by another F (abasic) site. Although we have not directly
addressed the possibility that this poor activity is due to prior
cleavage of the unlabeled DNA strand, the fact that the extent of
substrate cleavage in these assays was always
One key point addressed here was the
3`-exonuclease activity reported for mammalian AP endonucleases (Seki et al., 1991, 1992, 1993). Several other groups (Nes, 1980;
Kane and Linn, 1981; Sanderson et al., 1989; Chen et
al., 1991; Winters et al., 1994) failed to detect such an
activity, but employed different substrates and different reaction
conditions. We made an effort here to reproduce the assay of Seki et al.(1991) and were able to detect a very low level of
exonuclease activity that was stimulated when using their conditions.
This activity was sensitive to the nature of the reaction buffer and
was more pronounced under conditions of high osmolarity (0.25 M sucrose). It also appears that the detailed nature of the duplex
DNA substrate affects this activity. Since the exonuclease was detected
both for native Ape purified from HeLa cells and for recombinant
protein isolated by two different procedures from bacteria, it seems
unlikely to have arisen from a contaminant common to all these
situations; this conclusion was bolstered by immunological experiments.
The very weak intrinsic exonuclease of Ape (4 orders of magnitude lower
than the AP endonuclease) may instead reflect the homology of this
protein to more potent exonucleases from E. coli and Drosophila (Sander et al., 1993). Although the
biological relevance of such a weak activity is not obvious, it could
arise either from a small amount of modified (truncated?) Ape protein
or from some minor component of the heterogeneous poly(dA-dT)
substrate. A more puissant activity toward a more specialized DNA
structure could be significant biologically.
The powerful effects of
a 5`-phosphorothioate on cleavage by the Ape protein merit comment. The
simplest interpretation of this result is that this phosphate is
attacked by the enzyme during cleavage (Demple and Harrison, 1994). The
inhibitory effect of such a substitution is consistent with the
homology between Ape protein and exonuclease III (Eckstein, 1985). More
noteworthy is the stereospecific effect of the Sp isomer, which might
suggest a stereospecific attack by Ape. Structural studies of these
proteins are required to address this and other fundamental issues. The
recent report (Mol et al., 1995) of a crystal structure for
exonuclease III suggests that such information may soon be at hand.
Dashes
represent the identical sequence as for oligonucleotide 1 (sequences
2-11), oligonucleotide 12 (sequences 13 and 14), or
oligonucleotide 14 (sequences 15-18). Sequences for
oligonucleotides 5 and 6 are identical to the sequence 1, except for
the centrally located 5`-phosphorothioate F analogs, denoted Rp
Reactions containing
8-400 nM duplex DNA substrate were performed under
standard conditions (see ``Experimental Procedures'') for 5
min at 37 °C. Ape (35,500 Da) was present at a final concentration
of 100 pg/ml (2.8 pM). Values of K
3`-Exonuclease
activity was determined using a
3`-[
Enzymatic activity was determined as described
under ``Experimental Procedures'' with Ape present at final
concentrations of 0.1 and 1 ng/ml and with Ape activity within the
linear range. Results were calculated based on at least four
independent measurements and are expressed as rates normalized to the
F:C arrangement; means and standard deviations are shown.
We thank David M. Wilson (no relation) for assistance
with some of the experiments, Dr. Elena Hidalgo for helpful advice
during the purification of recombinant Ape, Dr. Richard A. O. Bennett
for input on the manuscript, the Cell Culture Center (Minneapolis, MN)
and Dr. Lynn Harrison for providing HeLa S3 cells, Dr. Gregory Verdine
(Harvard University) for pKEN2 and the XA90 strain, Drs. Mark R. Kelley
and James P. Carney (Indiana University Medical School) for the
pGEX-Ape construct, and Dr. Bernard Weiss (University of Michigan) for
supplying the dCTP deaminase used during substrate preparation.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
> 1000-fold lower than for F. In contrast,
the specificity constant (k
/K
) for E or P
of Ape purified from HeLa cells was only 5-8-fold lower than for
F. Positioning a phosphorothioate ester immediately 5` to F inhibited
Ape incision activity 20-fold (Rp isomer) or > 10,000-fold (Sp
isomer). Although Ape did not have detectable endonuclease activity
toward single-stranded substrates or unmodified double-stranded DNA,
the enzyme displayed a low level of 3`-exonuclease activity for duplex
DNA (<0.03% of its AP endonuclease activity), which was influenced
by the reaction conditions. The base positioned opposite F did not
dramatically affect the cleavage efficiency of Ape, but an F:F
arrangement was cleaved at approximately one-third of the efficiency of
F:C. A 3`-mismatch diminished P and E cleavage only slightly and F not
at all. A 5`-mismatch reduced the Ape cleavage rate 4-10-fold for
F and
100-fold for P and E. A series of substrates with F at
different positions along the oligonucleotide showed that Ape requires
4 base pairs 5` to the abasic site and
3 base pairs on the
3`-side. The implications of these results for substrate recognition by
Ape are discussed.
(
)sites in DNA
are formed by a variety of mechanisms that include spontaneous or
chemically-induced hydrolysis of the N-glycosylic bond and
enzymatic release of modified bases by DNA glycosylases (Loeb and
Preston, 1986; Wallace, 1988; Lindahl, 1993; Demple and Harrison,
1994). It has been estimated that
10,000 AP sites are generated
per mammalian cell/day by purine-glycosyl bond hydrolysis under normal
physiological conditions (Lindahl and Nyberg, 1972); other sources of
AP sites add to this burden. Abasic sites are potentially lethal and
mutagenic (Loeb and Preston, 1986). To defend against these
consequences, cells possess ``class II'' (hydrolytic) AP
endonucleases that cleave on the 5`-side of AP sites to produce 3`-OH
groups and 5`-deoxyribose 5-phosphate residues (Demple and Harrison,
1994). Release of the 5`-abasic residue and DNA repair synthesis and
ligation complete the repair process (Demple and Harrison, 1994).
-lyase) activities (Demple and Harrison, 1994), are
refractory to DNA polymerases. The ``class II'' AP
endonucleases also have 3`-repair diesterase activity specific for this
collection of lesions (Demple and Harrison, 1994).
1/200 of its AP endonuclease activity; Chen et
al., 1991). Thus, other human enzymes may be involved in removal
of 3` damages (Chen et al., 1991; Winters et al.,
1992). On the other hand, Ape displays a powerful hydrolytic AP
endonuclease activity about 10-fold greater than found for exonuclease
III (Kane and Linn, 1981; Chen et al., 1991). A detailed
understanding of the requirements of the Ape enzyme for substrate
recognition and cleavage is clearly desirable. We have approached this
goal by determining the activity of purified human AP endonuclease on a
variety of synthetic oligonucleotide substrates containing AP site
analogs.
Materials
The oligonucleotides employed in these
studies were synthesized as described previously (Takeuchi et
al., 1994) except for the oligonucleotide containing
2-(aminobutyl)-1,3-propanediol residue (Q), which was purchased from
Operon Technologies, Inc. (Emeryville, CA). Structures of abasic sites
are shown in Fig. 1, and the sequences of the
oligodeoxynucleotides are displayed in . Bacteriophage T4
polynucleotide kinase was purchased from New England Biolabs (Beverly,
MA). The [-
P]ATP (specific activity, 3000
Ci/mmol) and [
H]dTTP (specific activity,
70-90 Ci/mmol) were obtained from DuPont NEN. Exonuclease III was
purchased from Life Technologies, Inc. DNase I was obtained from Sigma.
Figure 1:
Structures of natural and synthetic
abasic sites. The equilibrium between the ring (AP, right) and open-chain (AP, left) forms of
deoxyribose favors the former. The other symbols are shown to the right of each moiety.
HeLa AP Endonuclease
Human Ape protein was
purified from HeLa S3 cell extracts essentially as described by Chen et al.(1991), except that the Affi-Gel blue step was omitted.
Ape enzyme activity was detected during the purification by monitoring
the release of radiolabeled 3`-phosphoglycolaldehyde residues from a
synthetic DNA substrate (Chen et al., 1991). The purified Ape
protein used here had an AP endonuclease activity of 5.3
10
units/mg of protein, determined using a synthetic
substrate (Levin and Demple, 1990). One unit of enzyme cleaves or
releases 1 pmol of damaged sites/min under standard conditions (Levin
and Demple, 1991; Chen et al., 1991).
Purification of Recombinant Ape Proteins
Two
plasmids were constructed to express Ape protein for purification from E. coli. pDW204 was generated by inserting a polymerase chain
reaction-generated fragment containing the Ape coding region into the EcoRI and BamHI restriction sites of the pKEN2 vector
(a gift from Dr. G. L. Verdine). Its expression in lacI strains is inducible by
isopropyl-1-thio-
-D-galactopyranoside under the control
of a tac promoter. pGEX-Ape (graciously provided by Drs. Mark
R. Kelley and James P. Carney) was constructed by inserting the Ape
coding sequence into a BamHI- and EcoRI-digested
pGEX-3X (Pharmacia Biotech Inc.) behind the tac promoter and
in frame with the glutathione S-transferase gene.
xth nfo-1::kan derivative of strain XA90) containing
pDW204 were diluted 1:100 into LB broth and grown for 2 h at 37 °C,
and isopropyl-1-thio-
-D-galactopyranoside was added to a
final concentration of 1 mM. After 4 h of shaking at 37
°C, the cells were harvested by centrifugation and resuspended in
50 mM Hepes-KOH, pH 7.5, 125 mM KCl, 10% glycerol.
The cells were lysed by agitation with glass beads in a
Mini-Bead-Beater (Biospec Products, Bartlesville, OK). After
centrifugation at 12,000
g, the soluble material was
applied to a column of phosphocellulose (Whatman P-11) equilibrated
with 50 mM Hepes-KOH, pH 7.5, 125 mM KCl, 10%
glycerol. The column was washed with several column volumes of
equilibration buffer, and the bound material was then eluted with a
linear gradient of KCl (125-600 mM). Fractions
containing full-length Ape protein (determined by immunoblotting;
Demple et al.(1991)) were pooled and concentrated using a
Centriprep 30 apparatus (Amicon, Inc., Beverly, MA). The
phosphocellulose pool was further fractionated on an AcA54 (Pharmacia)
gel filtration column equilibrated with 50 mM Hepes-KOH, pH
7.5, 150 mM KCl, 10% glycerol. Fractions were analyzed for AP
endonuclease activity and protein concentration (determined by
Coomassie Blue staining), and the active fractions were pooled to yield
``recombinant Ape.''
-D-galactopyranoside as described for
DW5281/pDW204. After collection and resuspension, cells were lysed by
sonication, and Triton X-100 was added to a final concentration of 1%.
Following centrifugation for 10 min at 12,000
g, the
supernatant was applied to a glutathione-Sepharose 4B (Pharmacia)
column equilibrated with 50 mM Hepes-KOH, pH 7.5, 150 mM KCl, 10% glycerol, 0.1 mM dithiothreitol. The column was
washed with equilibration buffer containing 1% Triton X-100,
Ape-glutathione S-transferase fusion protein was eluted with
10 mM reduced glutathione (Sigma), and the active fractions
were pooled. To release Ape from the hybrid protein, samples were
incubated with CaCl
(6 mM final concentration) and
Factor Xa (0.5 units; Pierce, Rockford, IL), and the cleavage was
performed overnight at 4 °C. The ``clipped'' protein was
applied to a small phosphocellulose column as described above and
eluted with buffer containing 400 mM KCl.
Preparation of Double-stranded DNA
Substrates
Oligonucleotides containing abasic sites were
5`-end-labeled by treatment with T4 polynucleotide kinase and
[-
P]ATP (Sambrook et al., 1989).
The DNA strand to be labeled was always in molar excess over the ATP.
The kinase was heat-inactivated by incubation at 90 °C for 2 min;
the reaction was placed immediately on ice, and an equimolar amount of
the complementary oligonucleotide was added. This mixture was heated at
56 °C for 10 min, and the two DNA strands were annealed, first at
room temperature for 1 h and then at 4 °C overnight.
Enzymatic Reactions
Reactions to measure incision
activity of Ape were performed in 10 µl of 50 mM
Hepes-KOH, pH 7.5, 50 mM KCl, 100 µg/ml bovine serum
albumin, 10 mM MgCl, 0.05% Triton X-100 (Chen et al., 1991) containing the indicated amounts of
double-stranded DNA substrate. After incubation at 37 °C with
various amounts of Ape protein for the indicated times, the reactions
were stopped by placing them on ice and immediately adding 5 µl of
formamide solution (Sambrook et al., 1989). The samples were
then heated at 65 °C for 5 min, and 3-µl aliquots were analyzed
on 20% polyacrylamide, 7 M urea gels run at 20 V/cm. After
electrophoresis, the gels were autoradiographed to locate the labeled
substrate and product oligodeoxynucleotides. The conversion of
substrate to product was calculated by excising the substrate and
product bands, counting the radioactivity in the gel slices in a
Beckman LS 1801 scintillation counter, and calculating fractional
conversion (fractional conversion = cpm in product/(cpm in
product + cpm in substrate)).
were performed as described
by Takeuchi et al.(1994).
Exonuclease Activity of Ape
To specifically
measure exonuclease activity of Ape, two methods were employed. For the
first method, the procedure described by Seki et al.(1991),
using 3`-end-labeled copolymers, was implemented. These experiments
were performed with various Ape protein preparations. In the second
method, poly(dA-dT) duplex DNA containing a P-radiolabeled
dUMP approximately every 600 nucleotides (Levin et al., 1991)
was treated with DNase I (see figure legends for concentration) for 2
min at room temperature in Ape AP endonuclease buffer (see
``Enzymatic Reactions'' of this section). DNase I was
subsequently inactivated by incubating for 5 min at 65 °C, and the
reaction was placed on ice. Approximately 1 pmol of the resulting DNA
substrate was then incubated with recombinant Ape produced in bacteria
or exonuclease III for 30 min at 37 °C. The DNA polymer was
precipitated on ice for 10 min by the addition of trichloroacetic acid
to a final concentration of 0.65 M. After centrifugation at
12,000
g, the free radiolabeled nucleotides in the
acid-soluble supernatant were quantified by liquid scintillation
counting. Immunoprecipitation of Ape protein with rabbit antiserum and
protein A-Sepharose beads (Sigma) was performed essentially as
described by Chen et al.(1991), except that the primary and
secondary incubations were shortened to 2 and 1 h, respectively.
Incision Kinetics of Ape on Synthetic AP Site
Analogs
Oligonucleotides containing synthetic AP sites
(ethanediol (E), propanediol (P), or furanyl (F) moieties) were
5`-end-labeled and annealed to the unlabeled complementary strand with
deoxycytosine positioned opposite the abasic site (sequences 1, 2, or 3
annealed to sequence 7; see ). Ape incision activity on the
resulting labeled 18-bp, double-stranded DNA substrates was estimated
by calculating the percent conversion of substrate (18 bp) to product
(9 bp) after separation by denaturing gel electrophoresis (see
``Experimental Procedures''). Time course experiments
revealed that Ape catalyzed incision of the three substrates at nearly
equal rates (Fig. 2; see below). The kinetic parameters for these
reactions were determined by varying substrate concentrations. These
studies revealed a <2.5-fold variation in the turnover number (k), while the specificity constants (k
/K
) for these
abasic sites varied only
8-fold (F > P > E), primarily due
to >10-fold range for apparent K
(). None of these sites was cleaved in
single-stranded DNA, and a 10-molar excess of unlabeled AP site
containing single-stranded DNA did not alter Ape incision rates for
duplex DNA substrates (data not shown).
Figure 2:
Incision products of Ape on duplex
oligodeoxynucleotides containing E, P, or F. Substrates (200
nM) were incubated with Ape (100 pg/ml) under standard
reaction conditions for the time indicated, and the reaction products
were analyzed on a 7 M urea, 20% polyacrylamide gel (see
``Experimental Procedures''). Uncleaved, single-stranded
substrate migrates as an 18-mer, while the incision products migrate as
9-mers.
We also explored the action
of Ape on a commercially available oligonucleotide substrate containing
a branched abasic site, a 2-(aminobutyl)-1,3-propanediol residue (Q,
sequence 4 in ; Fig. 1). In order to detect
Ape-mediated incision at Q, a concentration of enzyme was required that
was >1000-fold higher than needed for F (Fig. 3, lanes10 and 11). Similarly, concentrations of E.
coli exonuclease III that converted nearly all of the F-containing
substrate to product demonstrated very little endonucleolytic activity
on Q, although some exonuclease activity was observed (Fig. 3, lanes5 and 6). A 10-fold higher amount of
exonuclease III did not dramatically increase the amount of product
generated following incubation with Q (Fig. 3, lane7), but incision of Q by E. coli endonuclease IV
occurred at a rate only 8-fold slower than for F (Fig. 3, lanes3 and 4versuslane2).
Figure 3:
Activity of AP endonucleases on the
branched Q substrate. Duplex DNA substrates containing either F or Q
(100 nM) were incubated for 5 min with endonuclease IV,
exonuclease III, or Ape under standard reaction conditions in 10 µl
and analyzed by gel electrophoresis (see ``Experimental
Procedures''). Lane1, Q, no enzyme; lane2, F, 1 ng of endonuclease IV; lane3,
Q, 1 ng of endonuclease IV; lane4, Q, 10 ng of
endonuclease IV; lane5, F, 1 ng of exonuclease III; lane6, Q, 1 ng of exonuclease III; lane7, Q, 10 ng of exonuclease III; lane8,
Q, 1 ng of endonuclease IV; lane9, Q with no enzyme; lane10, Q, 1 ng of Ape; lane11,
Q, 10 ng of Ape. In lanes2-4 and 8 the upper bands are due to residual
substrate.
Exonuclease Activity for Ape
In previous studies
of Ape protein isolated from HeLa cells, we were unable to detect
significant 3`-exonuclease activity using an end-labeled, blunt-ended
duplex DNA substrate (Chen et al., 1991). However, it seemed
possible that the exonuclease activity reported for this protein by
others (Seki et al., 1991, 1992, 1993) could depend on the
substrate or reaction conditions. We therefore adopted the approach of
Seki et al.(1991) and investigated exonuclease activity of Ape
purified from HeLa cells and of recombinant protein isolated by two
different procedures from E. coli, either as intact Ape or as
the Ape segment liberated from a fusion with glutathione S-transferase (see ``Experimental Procedures'').
These experiments revealed a low but consistent amount of exonuclease
activity for all three forms of Ape (I). This activity
depended on the assay buffer, and was observed at levels
1.5-7-fold higher in the reaction buffer of Seki et al. (1991) than in our standard conditions.
Figure 4:
3`-Exonuclease activity of Ape protein. A, immunoprecipitation of Ape-specific exonuclease. Ape
purified from HeLa cells or the recombinant protein isolated from E. coli (expression vector pDW204; see text) was incubated
with the indicated amount of Ape-specific rabbit antiserum (Chen et
al., 1991) followed by the addition of protein A-Sepharose beads,
further incubation, and removal of the beads by centrifugation (see
``Experimental Procedures''). The resulting supernatants were
then assayed for 3`-exonuclease activity by the method of Seki et
al. (1991). Addition of >1 µl of serum was precluded by the
presence of exonuclease activity in the serum itself. B,
alternative exonuclease assay. Poly(dA-dT) containing one
[-
P]dUMP residue/
600 nucleotides was
treated with 0.1 ng (upperthreebars) or 1
ng (lowerthreebars) of DNase I (see
``Experimental Procedures''). This DNA was then incubated
with no protein, 3 AP endonuclease units of recombinant Ape protein, or
4 AP endonuclease units of exonuclease III. Following precipitation
with trichloroacetic acid, radioactivity in the supernatant was
determined by scintillation counting.
An alternative approach with a
different substrate revealed no significant exonuclease activity for
recombinant Ape protein under our standard reaction conditions (Fig. 4B). Consistent with the exonuclease constituting
a minor activity of Ape, the expected sizes of substrate and product
were observed in assays of Ape with oligonucleotide substrates (see Fig. 2, 3, and 6).
Inhibition by Phosphorothioate
Since the
phosphodiester cleaved by Ape is positioned immediately 5` to the
abasic site (Chen et al., 1991), we examined the effects on
Ape activity of substituting this phosphate with a phosphorothioate
residue (sequences 5 and 6 in ; Fig. 1). Ape actively
incised a duplex with an Rp phosphorothioate at a rate of approximately
one-twentieth of that seen for an F substrate with a conventional
phosphodiester (Fig. 5). Cleavage of the corresponding Sp isomer
was not detected (Fig. 5); the calculated cleavage rate of Ape
for the Sp isomer was <10 of that found for F.
Inhibition by 5`-phosphorothioates is consistent with this site as the
target for hydrolysis by Ape.
Figure 5:
Activity of Ape on phosphorothioate
substrates. Results show the incision rates of the phosphorothioate
substrates (RpF or Sp
F) compared with those for
E, P, or F containing normal phosphodiesters (see Fig. 2). Ape was
present at a concentration of 0.1 ng/ml (E, F, P) or 1 ng/ml
(Rp
F, Sp
F), and the reactions were performed as
described (see ``Experimental
Procedures'').
Influence of the Local DNA Structure on Ape Incision
Rates
The overall requirements of Ape for duplex DNA structure
around the cleavage target site were explored using a series of
substrates containing different bases in the opposing strand (sequences
12-18 in ) or base mismatches located in different
positions (sequences 1-3 and 8-11 in ), or by
varying the position of the abasic site along the duplex. The identity
of the base opposite an F residue did not significantly influence the
rate of cleavage by Ape (). However, a substrate
containing a F:F pair was incised at only approximately one-third of
the rate of substrates with a normal base in the opposite strand (). We have not determined to what extent this reduction
is due to cleavage of the unlabeled strand by Ape before the enzyme
interacts with the labeled strand. Preliminary experiments(
)indicate that such cleavage might be inhibitory.
4-fold compared with F in a fully base-paired context (). An adjacent 5`-mismatch had an even more pronounced
effect on the E or P sites, reducing the Ape incision activity
100-fold (). In contrast, a base mismatch on the
3`-side had only marginal effects on the cleavage rate for any of the
substrates. Mismatches on both the 5`- and the 3`-sides had an even
greater inhibitory effect on the Ape endonuclease activity than a
5`-mismatch alone, with the relative effect of the dual mismatch
particularly pronounced in the case of the F moiety ().
Thus, Ape seems to have a stronger requirement for duplex DNA structure
5` to the abasic site than it does for the 3`-side.
Figure 6:
Effect of the position of F within a
duplex oligodeoxynucleotide on the incision activity of Ape. Sequences
where F was placed at positions 1-17 (as indicated above each lane) throughout an 18-bp duplex DNA were incubated
with Ape (100 pg/ml) under standard reaction conditions and analyzed by
gel electrophoresis (see ``Experimental
Procedures'').
(Takeuchi et al., 1994). The
relatively robust activity of these enzymes against E (ethanediol
derivative) contrasts with the profile for E. coli endonuclease IV, for which E is a poor substrate (Takeshita et
al., 1987; Takeuchi et al. 1994). In a more limited
study, the bovine AP endonuclease homologous to exonuclease III (Robson et al., 1991) acted relatively efficiently on E (Sanderson et al., 1989). The opposite preference obtains for the
branched Q substrate, which is utilized poorly by Ape and exonuclease
III (Ca
) but effectively by endonuclease IV. These
preferences may be summarized as follows: the Ape/exonuclease III class
prefers abasic sites without branches at carbon 4 (equivalent to carbon
2 in Q; Fig. 1), but will tolerate distortions of phosphodiester
spacing implicit in the structure of E.
20% argues against
this interpretation. The poor activity of Ape on such a bistranded
substrate may underlie the biological effectiveness of antitumor drugs
such as bleomycin, which generate bistranded lesions in abundance
(Steighner and Povirk, 1990).
Table: List of oligodeoxynucleotides
and Sp
. Underlined letters indicate either the AP
site analog or the base positioned opposite the AP site.
Table: Kinetic parameters of Ape for duplex
DNA substrates containing synthetic AP sites
and k
were determined from
Lineweaver-Burk plots.
Table: Exonuclease
activities of native and recombinant Ape proteins
H]dTMP-labeled poly (dA-dT) substrate
prepared as described by Seki et al. (1991) but using either
the reaction buffer of Chen et al. (1991) as standard
conditions or that of Seki et al. (1991) as modified
conditions. AP endonuclease activity was assayed according to Levin and
Demple (1990) as modified by Chen et al. (1991). The isolation
of recombinant Ape proteins is described under ``Experimental
Procedures.'' AP endo, AP endonuclease activity; 3`-exo,
3`-exonuclease activity.
Table: Effect of the base positioned opposite F on
endonuclease activity
Table: Effects of flanking mismatches on endonuclease
activity
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.