The Exonuclease Activity of Human Apurinic/Apyrimidinic
Endonuclease (APE1)
BIOCHEMICAL PROPERTIES AND INHIBITION BY THE NATURAL
DINUCLEOTIDE Gp4G*
Kai-ming
Chou and
Yung-chi
Cheng
From the Department of Pharmacology, Yale University School of
Medicine, New Haven, Connecticut 06520
Received for publication, December 1, 2002, and in revised form, January 10, 2003
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ABSTRACT |
Human DNA apurinic/apyrimidinic endonuclease
(APE1) plays a key role in the DNA base excision repair process. In
this study, we further characterized the exonuclease activity of APE1.
The magnesium requirement and pH dependence of the exonuclease and endonuclease activities of APE1 are significantly different. APE1 showed a similar Km value for matched, 3'
mispaired, or nucleoside analog
-L-dioxolane-cytidine
terminated nicked DNA as well as for DNA containing a tetrahydrofuran,
an abasic site analog. The kcat for exonuclease
activity on matched, 3' mispaired, and
-L-dioxolane-cytidine nicked DNA are 2.3, 61.2, and 98.8 min
1, respectively, and 787.5 min
1 for APE1
endonuclease. Site-directed APE1 mutant proteins (E96A, E96Q, D210E,
D210N, and H309N), which target amino acid residues in the endonuclease
active site, also showed significant decrease in exonuclease activity.
Gp4G was the only potent inhibitor to compete against the
substrates of endonuclease and exonuclease activities among all tested
naturally occurring ribo-, deoxyribo-nucleoside/nucleotides, NAD+, NADP+, and Ap4A. The
Ki values of Gp4G for the endonuclease and exonuclease activities of APE1 are 10 ± 0.6 and 1 ± 0.2 µM, respectively. Given the relative concentrations of
Gp4G, 3' mispaired, and abasic DNA, Gp4G may
play an important role in regulating APE1 activity in cells. The data
presented here suggest that the APE1 exonuclease and AP endonuclease
are two distinct activities. APE1 may exist in two different
conformations, and each conformation has a preference for a substrate.
The different conformations can be affected by MgCl2 or
salt concentrations.
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INTRODUCTION |
DNA base excision repair is the main pathway to repair DNA base
damages caused by oxidation, radiation, and the loss of bases (1, 2).
There are several enzymes that participate in this pathway, including
DNA polymerase
, DNA ligase III-XRCC1 complex (1, 2), and
apurinic/apyrimidinic endonuclease
(APE1)1 to protect the genome
integrity (1-3). The DNA repair activity of APE1 resides in the
C-terminal region (4, 5). It endonucleolytically makes a nick
immediately adjacent to 5' of an apurinic/apyrimidinic (AP) site and
generates a hydroxyl group at the 3' terminus upstream of the nick and
a 5'-deoxyribose phosphate moiety downstream (6). DNA polymerase
further processes the product of APE1 by inserting a nucleotide into
the gap and releasing the 5'-deoxyribose phosphate by its intrinsic
lyase activity (7). The repair process is then completed by either DNA
ligase I or DNA ligase III/XRCC1 to seal the nicked DNA (1). During DNA
base excision repair, DNA polymerase
is the major polymerase that
incorporates a nucleotide into the gapped DNA (1). However, DNA
polymerase
is a lower fidelity polymerase (8) in comparison to the
replicating DNA polymerases
or
(9). The error rate of
polymerase
is about 1 in 4000 incorporations (1).
APE1 is a versatile multifunctional protein (10). In addition to its
endonuclease activity, it also possesses 3'-phosphatase, 3'-phosphodiesterase, RNase H, and 3'-5'-exonuclease activities (10).
APE1 knockout mice die in the early embryonic stage (11), which
indicates this protein is critical for development. Previously we
reported (12) that the 3'-5'-exonuclease activity of APE1 is the major
exonuclease activity in the human cell nucleus for the removal of the
nucleoside analog
-L-dioxolane-cytidine
(L-OddC, BCH-4556, Troxacitabine, and Troxatyl)
from the 3' termini of DNA. L-OddC is a novel nucleoside
analog with L-configuration that is currently under phase
III clinical trial and is showing promising activity for the treatment
of leukemia (13, 14). The incorporation of L-OddC into DNA
terminates DNA chain elongation because of the lack of the 3'-hydroxyl
group on the sugar moiety of L-OddC. The cytotoxicity of
L-OddC is proportional to the amount of L-OddC
in DNA, suggesting that the mechanism of action of L-OddC is to stop DNA replication. Therefore, the exonuclease activity of APE1
may play a critical role in determining the cytotoxicity of
L-OddC (15). We also discovered that APE1 has a significant preference for the removal of mispaired nucleotides from the 3' terminus of DNA when compared with matched pairs (16). Because physical
interaction between polymerase
and APE1 had been established (17)
and APE1 showed a significant preference for 3' mispaired nicked DNA
(16), APE1 could be the proofreading enzyme correcting the
misincorporations introduced by DNA polymerase
(16).
In addition to its DNA repair activities, APE1 was shown to possess
redox activity, which regulates the DNA binding of a number of
transcription factors, including Jun and Fos (18, 19). The redox
activity of APE1 resides in the N-terminal region (5), and cysteine 65 is considered to be the active site (4, 20). Previous reports (4, 20)
indicated that Cys-65 has no impact on the DNA endonuclease activity of
APE1. The biological function of this redox activity of APE1 is,
however, not well understood.
Several groups (21-23) have solved the crystal structure of APE1
in the presence or absence of abasic DNA. Mol et al. (22) reported the crystal structure of APE1-AP DNA complex and proposed a
mechanism of action for the APE1 endonuclease activity in which amino
acid Glu-96 binds to a metal ion, His-309/Asp-283 interacts with the
phosphate backbone of DNA, and Asp-210 deprotonates a water molecule
during nucleophilic attack on the AP site (22). Beernink et
al. (23) subsequently proposed another mechanism of action based
on the structure of APE1 crystallized under a neutral pH in the absence
of AP DNA. According to the latter model, there are two metal-binding
sites in the active center of APE1 endonuclease; Glu-96 is involved in
coordinating one metal ion, which stabilizes the O-3' leaving group
(23); and Asp-210 and His-309 coordinate the other metal ion to
deprotonate a water molecule, which generates a nucleophile. In both
models, Glu-96, Asp-210, and His-309 are involved in endonucleolytic
activity of APE1. Mutations at these sites resulted in a significant
decrease in endonuclease activity.
The monophosphate metabolites of ribonucleosides and several nucleoside
analogs have been shown to have inhibitory effects on DNA exonucleases
and DNA polymerase-associated exonucleases (24-27). In this study, we
have characterized the biochemical properties of APE1 including optimal
pH, Mg2+ requirements, and kinetic parameters of matched,
3' mispaired as well as L-OddC terminated DNA. The role of
the specific amino acids Cys-65, Glu-96, Asp-210, and His-309 in the
exonucleolytic action of APE1 was also examined. In addition, we have
examined the potential inhibitory effects of naturally occurring
ribonucleosides and deoxyribonucleosides and nucleotides on both
exonuclease and endonuclease activities of APE1. Interestingly, among
all the nucleosides and nucleotides, the dinucleotide compound
Gp4G was the only potent inhibitor of both endonuclease and
exonuclease activities of APE1. Gp4G is a product of
GTP:GTP guanylyltransferase (28, 29) or originates from the low
activity of mRNA capping enzymes (30). Although the detailed
functions of Gp4G are not well understood in the cell,
several Gp4G analogs were synthesized and shown to be
substrates for human immunodeficiency virus-reverse transcriptase (30,
31). The inhibition of APE1 activities by Gp4G suggests a
novel regulatory role of Gp4G for APE1 in cells.
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EXPERIMENTAL PROCEDURES |
Materials and Compounds--
[
-32P]ATP and
terminal deoxynucleotidyltransferase were purchased from Amersham
Biosciences. L-OddCTP was synthesized in our laboratory as
described (31). T4 polynucleotide kinase was purchased from New England
Biolabs (Beverly, MA). Gp4G was purchased from Sigma, and
purity (>95%) was confirmed by high pressure liquid chromatography.
Oligonucleotide Substrates--
All oligonucleotides were
synthesized by Integrated DNA Technology, Inc. (Coralville, IA), and
further purified by electrophoresis on a 20% denaturing polyacrylamide
gel. The 21-mer oligonucleotides were 5'-end-labeled with
[
-32P]ATP using T4 polynucleotide kinase.
The 32P-labeled 21-mer was then annealed to a 40-mer
oligonucleotide to generate a recessed DNA with or without a mispair at
the 3' terminus. To generate nicked mispaired and matched DNA, an
additional 19-mer oligonucleotide was added for the annealing reaction.
The sequences of the 3' mispaired recessed (Sequence 1) or nicked DNA
(Sequence 2) are shown.
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The sequence for the AP site analog (tetrahydrofuran, F) DNA
(F-DNA) is shown in Sequence 3,
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Site-directed Mutagenesis--
The pET-28b expression plasmid
containing wild type APE1 was a generous gift from Dr. Bruce Demple
(Harvard University). Mutagenesis was performed by the PCR-based method
using the QuickChange Site-directed Mutagenesis Kit (Stratagene). The
primers used to create each mutant form are as follows: APE1-C65A
primer, 5'-GCC ACA CTC AAG ATC GCC TCT TGG AAT
GTG GAT GGG; APE 1-E96A primer, 5'-ATA CTG TGC CTT CAA
GCG ACC AAA TGT TCA GAG AAC; APE 1-E96Q primer.
5'-ATA CTG TGC CTT CAA CAG ACC AAA TGT TCA GAG AAC; APE 1-D210E primer, 5'-CTT GTG CTG TGT GGA
GAG CTC AAT GTG GCA CAT GAA; APE 1-D210N primer,
5'-CTT GTG CTG TGT GGA AAC CTC AAT GTG GCA CAT
GAA; APE1-H309N primer, 5'-GCC CTC GGC AGT GAT
AAC TGT CCT ATC ACC CTA TAC.
The DNA sequences of the expression plasmids containing the wild type
or the site-directed mutant APE1 genes were confirmed by DNA sequencing at HHMI/Keck Facility, Yale University.
Purification of Recombinant APE1 and the Site-specific Mutant
HAP1 Proteins--
The wild type and mutant pET-28b-APE1 constructs
were expressed in Escherichia coli BL21(DE3) cells. The
cells were grown at 37 °C in a shaking incubator until the culture
reached an A600 of 0.6. Expression of
APE1 from the T7 promoter was induced for 3 h by addition of 1 mM isopropyl-1-thio-
-D-galactopyranoside (final concentration). The cells were then harvested and lysed by
sonication, and the cell debris was pelleted by ultracentrifugation (27,500 rpm, 4 °C, 40 min in a Beckman 28 Ti rotor). The supernatant was dialyzed against nickel column binding buffer (Buffer A: 20 mM Tris-HCl, pH 7.4, 0.5 M NaCl, and 20 mM imidazole) before loading onto a
Ni2+-charged His-trap fast protein liquid chromatography
column (Amersham Biosciences), and the bound protein was eluted with a
20-300 mM imidazole gradient in binding buffer. The
elution fractions that contained APE1 protein were pooled and dialyzed
against Buffer A (20 mM Tris-HCl, pH 7.4, 10% glycerol, 1 mM dithiothreitol, 1 mM EDTA, and 20 mM NaCl). The dialyzed sample was then applied to a 1-ml
fast protein liquid chromatography Hitrap SP XL column (Amersham
Biosciences). The bound protein was eluted with a linear gradient of
100% Buffer A to 100% Buffer B (20 mM Tris-HCl, pH 7.4, 10% glycerol, 1 mM EDTA, and 1 mM
dithiothreitol, 1 M NaCl).
The purified proteins were stored at
70 °C in storage buffer (20%
glycerol, 20 mM NaCl, 20 mM Tris-HCl, pH 7.4, 1 mM EDTA). All proteins were >95% pure as judged by
electrophoresis on 12% SDS-polyacrylamide gels stained with Coomassie
Blue (data not shown).
Enzyme Assays--
Standard reaction (10 µl) mixtures
contained 20 mM Tris-HCl, pH 7.5, 50 mM KCl,
0.1 mg/ml bovine serum albumin, optimal MgCl2, and various
concentrations of DNA substrates and enzymes. The reaction mixtures
were incubated at 37 °C for various times and then stopped by adding
4 µl of gel loading solution (90% formamide, 1 mM EDTA,
0.1% xylene cyanol, and 0.1% bromphenol blue) and heated at 80 °C
for 3 min. Samples (4 µl) were loaded onto a 20% denaturing polyacrylamide gel for electrophoresis. The gel was then dried under
vacuum and subjected to autoradiography and a PhosphorImaging screen
(Bio-Rad). For kinetic analysis, the concentration of DNA substrates
ranged from 1 to 500 nM at a fixed enzyme concentration. For each reaction, less than 25% of the substrates were converted to
products by the enzyme, even at the lowest substrate concentration. All
the kinetic data were determined based on the initial reaction rates.
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RESULTS |
The Optimal pH for the Exonuclease and Endonuclease Activities of
APE1--
Two different crystal structures of APE1 were obtained under
pH 6.2 and 7.5 (22, 23), which suggested that pH could cause conformational changes in APE1. We therefore determined the optimal pH
for both the endo- and exonuclease activities of APE1 under otherwise
identical reaction conditions. In addition, DNA substrates used shared
the same length and sequence context to eliminate the potential
influence of sequence context (see "Experimental Procedures"). As
shown in Fig. 1a, optimal
exonuclease activity was detected at pH 7.4 using a 3' mispaired nicked
DNA as substrate, and the activity decreased dramatically as the pH
increased. The optimal pH for endonuclease activity was observed
between pH 7.7-7.9 using DNA containing an abasic site analog,
tetrahydrofuran (F-DNA). Both activities decreased at pH 6.5 and
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Fig. 1.
The magnesium and salt requirements of
APE1 endonuclease and exonuclease activities. a, the
exonuclease and endonuclease activities at different pH values. Each
reaction contains either 50 nM mispaired or F-DNA substrate
and 48 pM APE1 under the standard assay conditions for 3 min at 37 °C under the pH value shown. The activities were
normalized with the maximal activities (100%) at the tested pH.
b, the profiles of APE endonuclease and exonuclease
Mg2+ dependence. Each reaction contained 50 nM
DNA substrate (3' mispaired recessed DNA, 3' mispaired nicked DNA, or
F-DNA), 48 pM APE1, and different concentrations of
MgCl2 at the standard reaction conditions for 5 min at
37 °C, pH 7.4. The activities were normalized with the maximal activities for each substrate at tested
MgCl2 concentrations. c, the impact of NaCl on
APE1 endonuclease and exonuclease activity. Each reaction contained 50 nM 3' mispaired nicked or F-DNA in the presence of 2 mM MgCl2, 20 mM Tris-HCl, pH 7.4, 48 pM APE1, and various concentrations of NaCl. The
reaction mixtures were incubated at 37 °C for 5 min. This figure is
representative of three independent experiments.
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Magnesium Requirement for the Endonuclease and Exonuclease
Activities of APE1--
Magnesium is required for the endonuclease
activity of APE1, but high concentrations of magnesium inhibit this
activity (32, 33). We have demonstrated previously (12) that magnesium
is also required for the exonuclease activity of APE1. The recent crystal structure of APE1 revealed that there are two divalent metal
ion-binding sites in the active center at neutral pH (7.5) (23), but
only one divalent metal ion was observed in the crystal structure of
the APE-DNA complex at pH 6.5 (22). This suggested that optimal
Mg2+ concentrations for the endonuclease activity might
vary under different assay conditions, e.g. pH. We therefore
determined the optimal Mg2+ concentration required for the
exo- and endonuclease activity at the same pH (7.4).
As shown in Fig. 1b, neither activity was observed in the
absence of magnesium. The maximal exonuclease activity was observed between 0.1 and 1.0 mM MgCl2 on a 3' mispaired
recessed DNA substrate. Interestingly, a higher concentration of
MgCl2 was required to reach the maximal exonuclease
activity for the removal of 3' mispair from a nicked DNA substrate.
Similar Mg2+ dependence profiles were observed while using
recessed or nicked DNA with L-OddC at the 3' terminus (data
not shown). In contrast to the exonuclease activity, the endonuclease
activity required a much higher magnesium concentration to reach its
optimal activity (10-15 mM) under the same assay conditions.
The higher magnesium requirement for optimal endo- as compared with the
exonuclease activity may be due to a need for higher ionic strength in
the former reaction. In other words, both activities require a
similarly low Mg2+ concentration (around 2 mM)
for chemical catalysis, and the additional contribution of the metal
(around 15 mM) to the endonuclease activity is to increase
the ionic strength of the solution. To address this issue, both
activities were monitored at 2 mM MgCl2 with increasing amounts of NaCl using either 3' mispaired nicked DNA or
F-DNA as substrates. As shown in Fig. 1c, similar
exonuclease activity was observed with 0-60 mM NaCl, but
this activity decreased significantly above 100 mM. In
contrast, the endonuclease activity increased in a
salt-dependent manner. The maximal endonuclease activity
was observed at 120 mM NaCl, the same activity as observed using 12.5 mM MgCl2 and 20 mM NaCl.
The endonuclease activity decreased dramatically when the NaCl
concentration exceeded 300 mM. Under a reaction mixture
containing 12.5 mM MgCl2, only an inhibitory
effect was observed with increasing concentrations of NaCl (data not
shown). Thus, the higher MgCl2 concentration for optimal
endonuclease activity did not appear to be a specific requirement
during endonucleolytic catalysis.
The Steady-state Kinetic Studies of APE1 Exonuclease--
Because
the magnesium and salt requirements for endonuclease and exonuclease
activities are different, the kinetic parameters of both activities
were studied under the optimal conditions for each activity of APE1. To
determine the kcat and Km
values, the exonuclease activity of APE1 was measured using varied
concentrations (1-500 nM) of either a recessed or a nicked
double-stranded DNA (see "Experimental Procedures"). Previous
reports (34) showed discrepancies in the Km values
of APE1 endonuclease; therefore, the kinetic properties of APE1
endonuclease activity on F-DNA were also examined for comparison. The
Km and kcat values are
summarized in Table I. The
Km values of APE1 exonuclease for recessed 3'
mispaired and L-OddC terminated DNA substrates were similar
(37.8 ± 3.6 and 41.3 ± 6.2 nM, respectively) and 2-3-fold higher than those for nicked 3' matched, mispaired, L-OddC terminated DNA or F-DNA (16.2 ± 2.8, 12.6 ± 1.3, 19.8 ± 2.5, and 10.7 ± 1.5 nM,
respectively). The AP endonuclease activity of APE1, which is
considered to be the primary function of APE1 in cells, showed the
highest kcat (787.5 min
1) using
F-DNA as substrate. The APE1 exonuclease activity had a higher rate for
the removal of L-OddC than the 3' mispaired nucleotides
from the 3' terminus of either recessed or nicked DNA with
kcat values for L-OddC terminated
and 3' mispaired nicked DNA of 98.8 and 61.2 min
1,
respectively. APE1 exonuclease showed the lowest activity on matched
nicked DNA (kcat 2.3 min
1), which
is about 35-fold less than that for the 3' mispaired DNA and 342-fold
lower than the endonuclease activity on F-DNA under corresponding
optimal reaction conditions.
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Table I
The kinetic parameter of APE1 DNA endonuclease and exonuclease
For endonuclease activity, the buffer contained 20 mM
Tris-HCl, pH 7.4, 12.5 mM MgCl2, 20 mM
NaCl, and 0.1 mg/ml bovine serum albumin. For the exonuclease activity
on recessed DNA and nicked DNA, the same buffer was used but the
MgCl2 concentrations were 0.1 and 2 mM,
respectively. The enzyme concentration used for the exonuclease
activity on 3' matched nicked DNA substrate was 100 pM. The
enzyme concentration for 3' mispaired or L-OddC terminated
recessed or nicked DNA was 11.1 pM. The enzyme
concentration for the endonuclease activity on F-DNA was 1.2 pM.
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The Roles of Specific Amino Acids Involved in the Exonuclease
Activity--
The different magnesium requirements, salt dependence,
and kinetic parameters for the endonuclease and exonuclease activities of APE1 suggested that amino acids involved in these two activities may
play different roles during the reactions. To examine whether the
exonuclease activity of APE1 shares the same amino acids in the active
center with endonuclease activity, specific site-directed APE1 mutants
of amino acids Glu-96, Asp-210, and His-309, which have been shown to
be critical for the endonuclease activity (10, 33) were generated. The
mutants were designed to probe the impact of both the electrostatic
charge and spatial effects of these amino acid residues (see Table
II). Cys-65, a key player in the redox
activity of APE1 (4, 20) was also mutated to examine its potential
involvement in the exonuclease activity of APE1. The specific
activities were obtained from the initial reaction rate of the wild
type, and mutant APE1 proteins on recessed and nicked DNA with a 3'
mispair (sequences are shown under "Experimental Procedures") as
well as F-DNA are presented in Table II. The endonuclease activity of
wild type APE1 is about 30-fold higher than the exonuclease activity on
3' mispair from a nicked DNA and 80-fold higher than from the recessed
DNA. Both endonuclease and exonuclease activities of Cys-65 mutant APE1
were similar to those of the wild type, which suggested that Cys-65 is
not involved in either activity. Glu-96 was shown to be important for
the magnesium coordination during APE1 endonuclease action (23). As
shown in Table II, the endonuclease activity of E96A decreased
105-fold as compared with the wild type APE1. The
exonuclease activity of E96A decreased about 104-fold on 3'
mispaired recessed DNA and 6,100-fold on nicked DNA. E96Q showed better
(~35-fold) exonuclease activity on both recessed and nicked 3'
mispaired DNA than E96A. The Mg2+ dependence profiles of
E96Q exonuclease activity were very similar to that of wild type, but
the endonuclease activity of E96Q showed no significant change under
different Mg2+ concentrations, although Mg2+
was required (Fig. 2a).
Moreover, the product of E96Q endonuclease activity only increased
slightly with time (Fig. 2b), which is not the pattern
observed with wild type or other APE1 mutant proteins. To explore the
interesting results of E96Q endonuclease activity, we performed a
mobility retardation assay in the presence or absence of magnesium
(Fig. 2c). The results showed that magnesium increased the
formation of E96Q-F-DNA complex (Fig. 2c, lane
5). Under the same conditions, no complexes formed using wild type
(Fig. 2c, lane 4) or other mutant APE1 proteins
(data not shown), which suggested that dissociation of E96Q from its
endonuclease product is very slow.
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Table II
The impact of amino acid mutations on the endonuclease and exonuclease
activities of APE1
The specific enzyme activities of wild type or mutant APE1 proteins
were determined by the initial reaction rates of their endonuclease and
exonuclease activities in the presence of 50 nM F-DNA,
mispaired recessed, or nicked DNA under their optimal reaction
conditions (same as Table I). The concentrations of wild type and C65A
mutant APE1 proteins used are the same as Table I. For E96A mutant
APE1, 10 and 1.1 nM protein were used in the exonuclease
and endonuclease assays, respectively. For E96Q mutant APE1, 10 nM enzyme was used for the endonuclease assay, and 3.3 and
1 nM protein were used for the 3' mispaired recessed and
nicked DNA substrates, respectively. A final concentration of 10 nM enzyme was used for the endonuclease and exonuclease
activity assays for D210E, D210N, and H309N mutant APE1.
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Fig. 2.
The endonuclease activity of E96Q mutant
APE1. a, the magnesium dependence of E96Q endonuclease.
Each reaction contains 50 nM F-DNA and 5 nM
E96Q mutant APE1 protein and increasing amounts of MgCl2,
ranging from 0 to 30 mM (lanes 2-11).
b, the time course study of E96Q endonuclease activity. Each
reaction contains 50 nM F-DNA, 12.5 mM
MgCl2, and 5 nM E96Q mutant APE1 protein for
various times (lane 1, control DNA; lanes 2-6,
2, 4, 6, 8, 10 min, respectively). c, the mobility
retardation study of E96Q mutant APE1 protein. Each reaction contains
10 nM F-DNA, wild type (10 nM), or E96Q (10 nM) mutant APE1 protein. The reaction mixtures were
incubated at 37 °C for 5 min followed by addition of 20% glycerol
before loading onto an 8% native PAGE gel for electrophoresis. The gel
was exposed to a PhosphorImager screen. Lane 1, control DNA;
lane 2, wild type APE with F-DNA in 5 mM EDTA;
lane 3, E96Q with F-DNA in 5 mM EDTA; lane
4, wild type APE with F-DNA in 12.5 mM
MgCl2; lane 5, E96Q with F-DNA in 12.5 mM MgCl2.
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D210E mutant APE1 protein showed similar reduction in both endonuclease
and exonuclease activities as the E96A mutant APE1 protein. D210N APE1
mutant protein also showed very low endonuclease and exonuclease
activities. The H309N mutant APE1 showed no detectable exonuclease
activity, and the endonuclease activity decreased more than
105-fold in comparison to the wild type. The double mutant
E96A/D210A showed no endonuclease and exonuclease activities,
which suggested that there is no compensatory effect between these two
amino acids (data not shown).
With the exception of E96Q, the magnesium requirement profiles of C65A,
E96A, and D210A were similar to those of the endonuclease or
exonuclease activities of the wild type APE1 (data not shown).
Inhibition of Endonuclease and Exonuclease Activities of APE1 by
Dinucleotide Gp4G--
The monophosphate metabolites of
purine ribonucleosides and some nucleoside analogs were shown to
inhibit DNA exonuclease activities (24-27). In this study, the
naturally occurring ribo- and deoxyribonucleosides and their mono-,
di-, and triphosphate metabolites, dinucleotide polyphosphate
Ap4A and Gp4G, as well as the reduced and
oxidized forms of NAD+ and NADP+ were examined
for their potential inhibitory effect on APE1. Among all the tested
compounds, only Gp4G showed significant inhibition of both
endonuclease and exonuclease activities of APE1 (Table III and Fig.
3a). The IC50
values of Gp4G on the APE1 endonuclease activity on a F-DNA
substrate and exonuclease activity on a 3' mispaired nicked DNA were
11 ± 1.1 and 1.2 ± 0.3 µM, respectively (Fig.
3b). To understand the nature of Gp4G inhibition
on both APE1 activities, kinetic analysis was performed, and the
results demonstrated that Gp4G was a competitive inhibitor
with respect to the DNA substrates for both endonuclease and
exonuclease activities of APE1. The Ki values were
1 ± 0.2 µM for the exonuclease and 10 ± 0.6 µM for endonuclease activity (Fig. 3, c and
d).
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Table III
The inhibitory effects of nucleoside and nucleotides on the
endonuclease and exonuclease activity of APE1
To monitor exonuclease activity, each reaction contains 20 nM 3' mispaired nicked DNA, 2 mM MgCl2,
20 mM Tris-HCl, pH 7.4, 11.1 pM APE1, and
tested nucleosides/nucleotides (5mM) for 5 min at 37 °C,
and the final concentrations of reduced/oxidized forms of NAD and NADP,
Ap4A, and Gp4G in the reaction were 0.5 mM.
For endonuclease activity, each reaction contains 20 nM
F-DNA, 12.5 mM MgCl2, 20 mM Tris-HCl,
pH 7.4, 1.2 pM APE1, for 3 min at 37 °C. The
concentrations of compounds tested were the same as used in the
exonuclease assay.
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Fig. 3.
The inhibition of exonuclease and
endonuclease activities of APE1 by Gp4G. a,
the inhibitory effects of dinucleotides Ap4A and
Gp4G. For exonuclease activity (lanes 1-4),
each reaction contains 20 nM 3' mispaired nicked DNA, 2 mM MgCl2, 20 mM Tris-HCl, pH 7.4, 11.1 pM APE1, and 0.5 mM Ap4A or
Gp4G (lane 1, control DNA; lane 2,
APE1 only; lane 3, APE1 and Ap4A; lane
4, APE1 and Gp4G). The reaction mixtures were
incubated at 37 °C for 5 min. For endonuclease activity (lanes
5-8), each reaction contains 20 nM F-DNA, 12.5 mM MgCl2, 20 mM Tris-HCl, pH 7.4, 1.2 pM APE1, and 0.5 mM Ap4A or
Gp4G. Lane 5, control DNA; lane 6,
APE1 only; lane 7, APE1 and Ap4A; lane
8, APE1 and Gp4G. b, the inhibition of
exonuclease activity (lanes 1-8) and endonuclease activity
(lanes 9-16). For exonuclease activity, the reaction
conditions are the same as described in a in the presence of
various concentrations of Gp4G (lane 1, control
DNA; lanes 2-8, 0, 0.01, 0.03, 1, 2, 3, and 10 µM, respectively). The reaction mixtures were incubated at 37 °C for 5 min. For endonuclease activity,
reaction conditions are the same as a in the presence of
various concentrations of Gp4G (lane 9, control DNA;
lanes 10-16, 0, 1, 3, 10, 20, 30, and 100 µM,
respectively). The results were analyzed by denaturing gel and exposed
to a PhosphorImager screen for quantitation. The IC50
represented is the concentration of Gp4G required to
inhibit 50% of the reaction. c, Gp4G
competitively inhibits exonuclease activity of APE1. The reaction
condition is the same as described in Table I in addition to various
concentrations of Gp4G (0-2 µM). The
Ki value (1 ± 0.2 µM) was
determined by the K under
different concentrations of Gp4G. d,
Gp4G competitively inhibits APE1 endonuclease activity. The
reaction conditions are the same as described in Table I in addition to
various concentrations of Gp4G (0-30 µM).
The Ki value (10 ± 0.6 µM) was
determined by the K under
different concentrations of Gp4G. This figure is
representative of three independent experiments.
|
|
APE1 exists as a monomer in the cell (10), and there is only one active
site found within in this enzyme (22, 23). Therefore, the
Ki values of a competitive inhibitor on both
endonuclease and exonuclease should theoretically be the same. Because
the optimal concentrations of MgCl2 for endonuclease and
exonuclease activities are different, it is possible that
MgCl2 may affect the Km and
Ki values. To address this issue, the
Km and Ki values of APE1
endonuclease activity were determined under the optimal
MgCl2 concentration for APE1 exonuclease activity (2 mM). The results indicated that both the Km and Ki values of endonuclease
activity under this condition were similar to those under higher
MgCl2 concentration (12.5 mM) (data not shown)
but kcat decreased, suggesting that the
MgCl2 has no significant impact on Km
and Ki values. In addition, the
Km value of APE1 exonuclease at 12.5 mM
MgCl2 did not show a significant change (<2-fold). The
Ki value of Gp4G on exonuclease, under
12.5 mM MgCl2, could not be obtained as the
long incubation time caused non-enzymatic breakdown of
Gp4G.
 |
DISCUSSION |
We demonstrated previously (12) that APE1 is the major exonuclease
that removes the unnatural L-configuration nucleoside analog, L-OddC, as well as other L-nucleoside
analogs from DNA. We subsequently discovered that the exonuclease
activity of APE1 has a significant preference for the removal of 3'
mispaired nucleotides from DNA over the matched ones (16). Because
physical interaction between APE1 and DNA polymerase
has been
established, this novel 3' mispair removal function of APE1 could
explain why the mutation rate is lower than expected, given that the
error rate of DNA polymerase
is 1 per 4000, and at least
104 abasic sites are generated per cell per day (1).
In this report, we have further characterized the biochemical
properties of the exonuclease activity of APE1. Several crystal structures of APE1 have been solved in the presence or absence of
abasic DNA under different crystallization conditions (21-23). Based
on the structures, different mechanisms of action were proposed for the
endonuclease activity of APE1. The latest crystal structure solved by
Beernink et al. (23) indicated that there are two divalent
ion binding sites in the APE1 active center under neutral pH. Given the
fact that the optimal pH for APE1 endonuclease activity is between 6.6 and 8.6 (23), the crystal structure solved under neutral pH could be a
more favorable conformation during the endonuclease action of the
enzymes. However, because the structure was crystallized in the absence
of DNA, the structure of APE1 bound to abasic DNA at neutral pH is
still not clear. As different structures of APE1 were obtained under
different pH, the optimal pH for the exonuclease activity of APE1 was
examined and compared with that of the endonuclease activity. The
results indicated that pH 7.4 is optimal for the exonuclease activity
as well as for the endonuclease activity, and this observation agrees
with a previous report (23).
Both endonuclease and exonuclease activities require magnesium for the
catalytic reactions (12, 33). At pH 7.5, the optimal magnesium
requirement for exonuclease activity (0.1-2 mM) is
substantially lower than that of endonuclease (10-15 mM).
Interestingly, as shown in Fig. 1c, at a low
MgCl2 concentration (2 mM) NaCl could achieve
the same endonuclease activity as the higher concentration of
MgCl2 (12.5 mM). This suggests that both
endonuclease and exonuclease activities of APE1 may have the same
MgCl2 requirement; however, the higher MgCl2
concentration requirement for the optimal endonuclease activity may not
be specific because NaCl can achieve the same activity. The crystal
structure showed that the abasic DNA was severely distorted from B-form
DNA during the endonuclease action (22). Although the
Km values of APE1 exonuclease on 3' mispaired nicked
DNA and endonuclease on F-DNA are very similar, despite different
MgCl2 concentrations (2 and 12.5 mM,
respectively), the structure of nicked DNA is very different from
AP-DNA (no strand breakage). Therefore, the higher concentrations of
MgCl2 or NaCl may facilitate the enzyme conformation
changes to favor its action on F-DNA.
The Km of APE1 exonuclease on recessed DNA is about
3-fold higher than on the nicked or F-DNA, which suggested that APE1
could have higher affinity for nicked or F-DNA than the recessed DNA.
It also explains the higher efficiency of APE1 in the removal of 3'
mispaired nucleotide from nicked than the recessed DNA (16), although
the kcat values are similar for both substrates.
In contrast, the kcat value of exonuclease on
the matched DNA is significantly lower than for either 3' mispaired or
L-OddC DNA. These data further support our previous results
(16) that APE1 has a higher efficiency for the removal of 3' mispaired
nucleotides than matched nucleotides from DNA. DNAs with
L-OddC at the 3' terminus were better substrates for the
APE1 exonuclease activity as compared with the 3' mispaired DNAs,
because the kcat values are higher than those
for the 3' mispaired DNA on both recessed and nicked DNA. The unnatural
L-configuration of L-OddC may cause a
distortion in DNA rendering it a more favorable substrate. Under the
optimal reaction conditions, F-DNA is the most favorable substrate for
APE1, and the kcat value of endonuclease activity was 9- and 6-fold higher than the exonuclease activity on the
3' mispaired or L-OddC terminated nicked DNA, respectively. More detailed pre-steady-state kinetic parameters are being explored to
further the understanding of the interactions between APE1 and various
DNA substrates as well as the catalysis of endonuclease and exonuclease cleavage.
The crystal structure of APE1 revealed that there is only one active
center in APE1 (21-23), although different mechanisms of action for
the endonuclease have been proposed. The redox activity of APE1 resides
within the N-terminal region, and Cys-65 has been implicated to be the
active center (4, 5, 19). The site-directed Cys-65 mutant APE1 protein
was shown to possess similar endonuclease activity as wild type (4,
20). In this report, we further showed that C65A mutant APE1 has
similar exonuclease activity as the wild type APE1 on both recessed and
nicked 3' mispaired DNA. These data further support that Cys-65 is not
involved in the DNA repair activity of APE1.
All the crystal structures of APE1, obtained under various
crystallization conditions, indicated that amino acids Glu-96, Asp-210,
and His-309 were critical for the endonuclease activity (21-23). The
site-directed mutagenesis results shown in Table II indicated that
mutation of Glu-96 to alanine reduced both exonuclease and endonuclease
activities of APE1 significantly (104 and 105,
respectively), which suggested that Glu-96 was critical but might have
a different impact on the two enzyme activities. The exonuclease
activity of E96Q decreased about 230-fold as compared with that of the
wild type APE1 (but was ~35-fold higher than that of E96A), which
indicated that the carboxyl group of Glu-96 might be critical for both
enzyme activities. Moreover, E96Q endonuclease activity did not require
higher MgCl2 concentration. Mobility shift studies using
native gel electrophoresis showed the E96Q mutant but not the wild type
or other mutant APE1 proteins bound to F-DNA in the presence of
magnesium (12.5 mM). To explore whether E96Q APE1 and F-DNA
formed covalent bonds, the complexes were boiled in the presence of SDS
and electrophoresed on an SDS-PAGE gel. No complexes were observed
after electrophoresis (data not shown), which indicated that no
covalent bonds formed in the complex. These results suggested that the
dissociation of E96Q mutant from its AP endonuclease product is very
slow, and Glu-96 is critical in determining the
kcat value for APE1 endonuclease activity. Glu-96 was proposed to play important roles in magnesium placement, and
the lack of dose dependence for magnesium and the slow dissociation of
E96Q from its DNA product suggested that the higher ionic strength requirement for the optimal APE1 endonuclease activity is needed for
the enzyme to disassociate from its product.
The APE1 D210E mutant protein also showed a significant decrease in
both endonuclease and exonuclease activities, although the substituted
glutamic acid can provide the same carboxyl group as the aspartic acid.
This indicated that steric hindrance has a profound effect on the
enzyme reaction. The D210N mutant APE1 showed extremely low exonuclease
and endonuclease activities, which supported the critical role of
carboxyl group of glutamic acid (35). The H309N mutant showed very low
exonuclease and endonuclease activities. Interestingly, the Glu-96 and
Asp-210 APE1 mutant proteins, with the exception of E96Q, showed
similar magnesium requirement profiles for endonuclease and exonuclease activities as the wild type APE1 on 3' mispaired recessed DNA, nicked
DNA, and F-DNA. We conclude that all the amino acids studied in this
report are important for both endonuclease and exonuclease activities
of APE1, but their roles in the two reactions may not be identical.
AMP and GMP were shown to be inhibitors for a number of DNA
polymerase-associated exonuclease activities (24-27, 36). Here we
report that the APE1 endonuclease and exonuclease activities cannot be
inhibited by AMP, GMP, and other natural nucleoside or their mono-,
di-, and triphosphate metabolites. Interestingly, Gp4G but
not Ap4A competitively inhibited both activities of APE1. The intracellular concentration of Gp4G in mammalian cells
is not clear yet; however, the physiological concentrations of
Gp4G was shown to be in the nanomolar to micromolar range
in rat tissues and human blood platelets (40, 41). In addition, the
concentrations of Gp4G were in the nanomolar to micromolar
ranges in Saccharomyces cerevisiae and E. coli
cells, depending on the growth condition (37). In exponentially growing
S. cerevisiae cells, the Gp4G concentration was
determined to be 60 nM, whereas after the temperature shift
to a non-growing condition, the concentration of Gp4G
increased to 1.15 µM (37). This implies that the
concentration of Gp4G may be higher in nonproliferating
tissues in mammals. In Artemia salina, Gp4G
comprises as high as 50% of the total nucleotide pool (10 mM) in dormant cysts, which was suggested to serve as a
source of purine for DNA synthesis (38). It was also proposed that
Gp4G regulates the activity of endoribonuclease VI in
Artemia, by alternating its concentration under different
developmental stages (39). An in vitro experiment has shown
that Gp4G activates GMP reductase activity in both
Artemia and human erythrocytes at nanomolar concentrations
(41, 42). Grau et al. (43) proposed a relationship between
Gp4G binding capacity and the time of development of rat
embryos. It was also suggested that Gp4G might play a role in the regulation of organ perfusion and vascular growth (40).
Interestingly, Gp4G can also be used as a primer for
template-directed synthesis of virus-specific oligonucleotides and RNA (30, 44). Furthermore, Gp4G can be synthesized by some
viral mRNA guanylyltransferases when RNA capping is uncoupled from
methylation and elongation (30). Several carbocyclic and 2'-deoxy
analogs of Gp4G were shown to be substrates for human
immunodeficiency virus-reverse transcriptase (30, 31), which suggested
that Gp4G may also affect viral replication. Given the
specificity and potency reported in this study and potential different
concentrations of Gp4G in different intracellular
compartments as suggested by others (38, 39), it is conceivable that
Gp4G may play important roles in regulating the enzyme
activities of APE1 in the cell nucleus.
In summary, this report reveals that APE1, in addition to its most
preferable endonuclease activity on abasic sites, also has very good
activity on 3' mispaired DNA and DNA terminated with the unnatural
L-configuration nucleoside analogs. Both endonuclease and
exonuclease activities of APE1 may have similar active centers, but the
amino acids involved may play different roles during each reaction.
MgCl2 is required for both activities, but higher ionic strength is required for endonuclease activity. It is possible that
higher ionic strength induces a change to a endonuclease-favored conformation. The different Ki values of
Gp4G on the two activities further supported the hypothesis
that APE1 may have two different conformations in equilibrium, and the
conformations can be affected by higher concentrations of
MgCl2 or NaCl. Gp4G may have different affinity
to these two forms; one form has a preference for the exonuclease
activity (lower ionic strength), and the other form favors endonuclease
activity (higher salt and magnesium requirement). We are currently
solving the co-crystal structures of APE1 with 3' mispaired nicked DNA
under different conditions to address this question.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Wing Lam, Zafer Hatahet, and
Adrian S. Ray for valuable discussions.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants CA 73477 and AI 38204.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: Dept. of Pharmacology,
Yale University School of Medicine, 333 Cedar St., SHM B315, New Haven,
CT 06520. Tel.: 203-785-7119; Fax: 203-785-7129; E-mail:
cheng.lab@yale.edu.
Published, JBC Papers in Press, March 6, 2003, DOI 10.1074/jbc.M212143200
 |
ABBREVIATIONS |
The abbreviations used are:
APE1, human
apurinic/apyrimidinic endonuclease;
L-OddC,
-L-dioxolane cytidine;
Gp4G, P1,P4-di(guanosine-5') tetraphosphate;
Ap4A, P1,P4-di(adenosine-5')
tetraphosphate;
F-DNA, DNA containing a tetrahydrofuran;
AP, apurinic/apyrimidinic..
 |
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