From the Department of Pathology and the Kaplan
Comprehensive Cancer Center, New York University School of Medicine,
New York, New York 10016, the § Department of Chemistry,
University of Connecticut, Storrs, Connecticut 06269, and the
¶ Department of Biological Sciences, The University at
Albany, State University of New York, Albany, New York 12222
Received for publication, December 1, 2002, and in revised form, January 7, 2003
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ABSTRACT |
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Base excision repair of oxidized
pyrimidines in human DNA is initiated by the DNA
N-glycosylase/apurinic/apyrimidinic (AP) lyase,
human NTH1 (hNTH1), the homolog of Escherichia coli
endonuclease III (Nth). In contrast to Nth, the DNA
N-glycosylase activity of hNTH1 is 7-fold greater than its
AP lyase activity when the DNA substrate contains a thymine glycol (Tg)
opposite adenine (Tg:A) (Marenstein, D. R., Ocampo, M. T. A., Chan, M. K., Altamirano, A., Basu, A. K.,
Boorstein, R. J., Cunningham, R. P., and Teebor, G. W. (2001) J. Biol. Chem. 276, 21242-21249). When Tg is
opposite guanine (Tg:G), the two activities are of the same specific
activity as the AP lyase activity of hNTH1 against Tg:A (Ocampo,
M. T. A., Chaung, W., Marenstein, D. R., Chan, M. K., Altamirano, A., Basu, A. K., Boorstein, R. J.,
Cunningham, R. P., and Teebor, G. W. (2002) Mol. Cell.
Biol. 22, 6111-6121). We demonstrate here that hNTH1 was
inhibited by the product of its DNA N-glycosylase activity
directed against Tg:G, the AP:G site. In contrast, hNTH1 was not as
inhibited by the AP:A site arising from release of Tg from Tg:A.
Addition of human APE1 (AP endonuclease-1) increased dissociation of hNTH1
from the DNA N-glycosylase-generated AP:A site, resulting
in abrogation of AP lyase activity and an increase in turnover of the
DNA N-glycosylase activity of hNTH1. Addition of APE1 did
not abrogate hNTH1 AP lyase activity against Tg:G. The stimulatory
protein YB-1 (Marenstein et al.), added to APE1, resulted
in an additive increase in both activities of hNTH1 regardless of base
pairing. Tg:A is formed by oxidative attack on thymine opposite
adenine. Tg:G is formed by oxidative attack on 5-methylcytosine
opposite guanine (Zuo, S., Boorstein, R. J., and Teebor, G. W. (1995) Nucleic Acids Res. 23, 3239-3243). It is
possible that the in vitro substrate selectivity of
mammalian NTH1 and the concomitant selective stimulation of activity by APE1 are indicative of selective repair of oxidative damage in different regions of the genome.
Like its Escherichia coli homolog, endonuclease III
(Nth), hNTH11 is a
bifunctional DNA N-glycosylase/apurinic/apyrimidinic
(AP) lyase that removes ring-saturated pyrimidines, be they hydrated, reduced, or oxidized, from the DNA backbone as the initial step of base
excision repair (BER) of such modified residues (1). The oxidation
product of thymine, 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol
(Tg)) is a widely studied model substrate for hNTH1 and catalyzes its
release from DNA via its DNA N-glycosylase activity. This
enzyme activity is mediated via formation of a transient Schiff base
(imino) enzyme-DNA intermediate, an example of covalent
catalysis and a characteristic of all known bifunctional DNA
N-glycosylases/AP lyases (4, 5). The Schiff base moiety has
been hypothesized to be required for the enzymatic catalysis of the
Based on the results of studies with Nth, the two activities of hNTH1
(DNA N-glycosylase and In our studies of the properties of hNTH1, we have focused our
attention on protein modulators of hNTH1 activities. This approach resulted from our original observation that the enzyme properties of
mammalian endonuclease III homologs are strikingly different from those
of bacterial endonuclease III. Our laboratory was the first to identify
mammalian endonuclease III homologs and to describe their distinct
kinetic properties (9, 10). These observations spurred us to isolate
novel proteins that might modulate the enzyme activities of mammalian
NTH1. A yeast two-hybrid search resulted in identification of the
pluripotent transcription factor YB-1 (Y-box-binding protein-1; also
known as DNA-binding protein B) as a stimulator of hNTH1 activity
(1).
We now report additional aspects of the proteomics of hNTH1-initiated
BER based on the effects of human APE1 (AP endonuclease-1; HAP1/REF1/APE) on hNTH1
activity. APE1, a human homolog of E. coli exonuclease III
(Xth), catalyzes the hydrolysis of the phosphodiester bond 5' to AP
sites, generating a free 3'-hydroxyl end for the initiation of DNA
polymerase repair synthesis.
APE1 has recently been demonstrated to play a significant role in the
coordination of BER. APE1 has been shown to modulate the activities of
several major DNA N-glycosylases, although physical interaction between APE1 and most of these DNA
N-glycosylases has not been demonstrated (6, 7, 11, 12). The
functional interaction between APE1 and DNA N-glycosylases
is consistent with the model of the coordinated activities of BER
enzymes at sites of DNA damage (13-16). Two groups independently
demonstrated the stimulation of human OGG1 (hOGG1) by APE1 (6, 7). Both groups demonstrated that APE1 replaces hOGG1 bound to the AP site following base release and prior to hOGG1-catalyzed Proteins--
Expression and purification of recombinant hNTH1
were induced as described previously (10). hNTH1 concentration was
quantified using an extinction coefficient of 1 absorbance unit
at A410 of 69.4 µM for the
C-terminal cubane 4[Fe-S] cluster (17). YB-1 was expressed with a
His6 tag in Trichoplusia ni cells and
purified as described (1). DNA polymerase 2'-Deoxyribose Oligonucleotides--
The 30-mer 2'-deoxyribose
oligonucleotide substrate containing a Tg residue at position 13 (d(GATCCTCTAGAGTgCGACCTGCAGGCATGCA)) was prepared as
described (1). The 30-mer 2'-deoxyribose oligonucleotide substrate
containing a uracil residue at position 13 of the identical sequence
and the complementary 2'-deoxyribose oligonucleotides were synthesized
by the Department of Cell Biology of the New York University School of
Medicine. 2'-Deoxyribose oligonucleotides were deblocked, deprotected,
and purified by 20% denaturing PAGE. The 2'-deoxyribose
oligonucleotides were gel-purified and labeled at the 5'-end using
[ Enzyme Assays--
Unless otherwise indicated, assays were
performed at 37 °C in buffer containing 50 mM HEPES (pH
7.6), 100 mM KCl, 0.1 mg/ml bovine serum albumin, 5 mM MgCl2, and 1 mM dithiothreitol.
Enzyme, protein, and substrate were diluted to working conditions in
assay buffer and equilibrated at 37 °C.
MgCl2-independent assays were performed with the above
reaction buffer minus MgCl2 in the presence of 5 mM EDTA, which permits binding of APE1 to DNA, but inhibits its endonuclease activity (18). Reactions contained 40 nM
32P-5'-end-labeled 2'-deoxyribose oligonucleotide
duplex substrate and 5 nM hNTH1 with or without 20 nM APE1. Two sets of 10-µl aliquots were taken at the
indicated time periods and snap-frozen in ethanol and dry ice, after
which one set was treated with 5 µl of 0.5 M putrescine
(pH 8.0) to measure base release. The treated assay mixtures were then
heated at 95 °C for 5 min, followed by addition of 15 µl of
loading dye (95% deionized formamide, 10 mM EDTA, 0.05%
bromphenol blue, and 0.05% xylene cyanol). To measure strand cleavage,
samples were treated with an equal volume of loading dye. All samples
were then heated at 55 °C for 5 min, and products were separated by
20% PAGE in 7 M urea and 1× Tris borate/EDTA. Low
substrate concentration assays were performed with 20 nM
hNTH1 and 20 nM 32P-5'-end-labeled
2'-deoxyribose oligonucleotide duplex substrate. At the indicated
times, aliquots were taken and treated as described above.
Multiple-turnover assays at Vmax were performed
individually in 10-µl volumes containing reaction buffer, the
indicated concentration of 32P-5'-end-labeled
2'-deoxyribose oligonucleotide duplex substrate, and 20 nM
hNTH1 with or without 100 nM YB-1 and/or 100 nM
APE1. The reactions were split into two 5-µl aliquots, snap-frozen in ethanol and dry ice, and treated as described above.
To discriminate between endonucleolytic and AP lyase products,
reactions were performed using 2'-deoxyribose oligonucleotide duplex
substrate labeled at the 3'-end with [
Single-turnover assays contained 10 nM 2'-deoxyribose
oligonucleotide duplex labeled at the 3'-end with
[ Cross-linking of Enzyme to 2'-Deoxyribose Oligonucleotide
Substrate (Enzyme Trapping)--
For NaBH4 reduction,
assays contained the indicated 32P-5'-end-labeled
2'-deoxyribose oligonucleotide duplex (400 nM) and 20 nM hNTH1 with or without 100 nM APE1 and/or 100 nM YB-1 in a volume of 10 µl. After a 15-min incubation,
reaction mixtures were treated with 5 µl of 0.5 M
NaBH4 and incubated at 37 °C for 5 min. After addition
of an equal volume of Laemmli SDS loading buffer, samples were boiled
in preparation for separation and analysis. All samples were separated
by 12% SDS-PAGE and analyzed quantitatively via phosphorimaging as
described above.
Differential Processing of Tg Opposite Adenine and Tg Opposite
Guanine by hNTH1--
We previously demonstrated, using a
Tg:A-containing substrate, that the initial rate of DNA
N-glycosylase-mediated release of Tg by hNTH1 is much
greater than the rate of AP lyase-mediated DNA strand cleavage (1).
Because all previous assays with Tg have been done with adenine as the
opposite base and because this may not be the only context in which Tg
occurs in vivo, we decided to look at hNTH1 activity against
substrates containing Tg paired opposite guanine (2). Fig.
1 is confirmation of our previous data.
The assay was carried out in a shorter reaction period than we
previously used and revealed that the difference between the rate of
base release and strand cleavage of a Tg:A substrate is even greater
than we had reported, differing by an order of magnitude. In sharp
contrast, the AP lyase activity of hNTH1 was the same as its DNA
N-glycosylase activity against a Tg:G substrate. The DNA
N-glycosylase activity of the enzyme against Tg:A appeared to reach Vmax at substrate concentrations >400
nM. However, as we previously reported, hNTH1 does not
follow Michaelis-Menten pseudo zero-order kinetics (1). Instead, the
apparent kcat (which includes the product
release rates) increased with increasing hNTH1 concentrations,
suggesting positive cooperativity in hNTH1 substrate processing. This
observation and interpretation have been corroborated and expanded upon
by Liu and Roy (20). Because of this phenomenon,
kcat and Km values for the
2'-deoxyribose oligonucleotide duplex substrates vary with enzyme
concentration. As we previously suggested, the V
versus S data of Fig. 1 reflect the fact that the enzyme was
dissociating from the DNA N-glycosylase-generated AP:A site
and initiating base release on new Tg:A substrates without catalyzing
APE1 Increases hNTH1 Processing of Tg:A-containing 2'-Deoxyribose
Oligonucleotide Substrates--
We looked at the effect of APE1 on the
activities of hNTH1. There is evidence that APE1 interacts with DNA
N-glycosylases in BER as part of a "single-nucleotide BER
relay" involved in the coordination of processing of BER pathway
intermediates (13, 21, 22).
Fig. 2 shows that addition of APE1
increased hNTH1 activity against a Tg:A substrate. The stimulation of
hNTH1 activity was independent of the enzyme activity of APE1. The
assay mixture lacked MgCl2 and contained sufficient EDTA to
abrogate APE1-mediated endonucleolytic cleavage, but not the binding of
APE1 to DNA (18). Identical experiments with Tg:G substrates showed no
increase in hNTH1 substrate processing in the presence of APE1 (data
not shown). We found that the optimum ratio for stimulation of hNTH1 by
APE1 varied with both hNTH1 and substrate concentrations, ranging from
1:1 to 1:10. We suggest that this variability was due, at least in
part, to the positive cooperativity exhibited by hNTH1 (1, 20).
APE1 Abrogates hNTH1 AP Lyase Activity on Tg:A-containing
Substrates, but Not on Tg:G-containing Substrates--
Having
determined that the effect of APE1 on hNTH1 activity differed with
substrate, we characterized the incision products generated by APE1 and
hNTH1 with the two substrates. The incision products generated by hNTH1
in the presence of APE1 in vitro differed with each
substrate. Fig. 3 is the gel analysis of
the incision products of 32P-3'-end-labeled Tg:A- and
Tg:G-containing 2'-deoxyribose oligonucleotide duplex substrates
generated by hNTH1 in the presence or absence of APE1. In contrast to
the experiments shown in Fig. 2, MgCl2 was added to the
reaction to activate the endonucleolytic activity of APE1. The
2'-deoxyribose 5'-phosphate residue generated by APE1-catalyzed
endonucleolytic cleavage of an AP site is extremely labile and not
retained during electrophoresis. Reduction by NaBH4 prior
to electrophoresis stabilizes the sugar moiety and permits size
discrimination between 5'-endonucleolytic and 3'- Incision Products Generated by hNTH1 in the Presence or Absence of
APE1, YB-1, and DNA Polymerase
Because hNTH1 did not demonstrate AP lyase activity against Tg:A sites
in the presence of APE1, we investigated whether the removal of the
2'-deoxyribose 5'-phosphate residue could be effected by DNA polymerase
APE1 Does Not Increase the DNA N-Glycosylase Activity of hNTH1
under Conditions of Single Turnover--
To elucidate the mechanism by
which APE1 affects hNTH1 activity, we investigated whether APE1 changed
the catalytic properties of hNTH1 or whether it affected product
inhibition. To address this, we conducted the reaction under
single-turnover conditions in which [E]
Fig. 5 illustrates that no stimulatory
effect of DNA N-glycosylase activity was observed under
conditions in which neither enzyme turnover nor substrate binding
contributed significantly to the rate of the reaction. A complete
abrogation of the AP lyase-mediated AP site cleavage was seen with the
Tg:A substrate (Fig. 5A). This is consistent with the
results of Figs. 3 and 4 (lanes 3).
Interestingly, in the case of the Tg:G substrate, addition of APE1
increased the rate of hNTH1 AP lyase activity (Fig. 5B). We previously mentioned that addition of APE1 did not result in increased product formation by hNTH1 under turnover conditions using
Tg:G (see above). Thus, the results of Fig. 5B suggest that hNTH1 remained tightly bound to the nicked AP:G DNA, which is the
product of AP lyase activity.
With both Tg:A and Tg:G substrates, the binding to the substrate and
base release were rapid, whereas APE1 Stimulates hNTH1 Activity under Conditions of
Vmax--
To determine whether APE1 increased the maximal
rate of turnover for the enzyme, we tested stimulation by APE1 under
conditions of Vmax for both DNA
N-glycosylase and AP lyase activities using 20 nM hNTH1 and 400 nM Tg:A substrate, identical
to the conditions of Fig. 1. However, in the experiments of Fig.
7, MgCl2 was added to
activate APE1 endonucleolytic activity. In reactions containing hNTH1
alone or with YB-1, DNA cleavage was due solely to hNTH1-catalyzed
Corroborating our results of experiments performed under low Tg:G
substrate concentrations, addition of APE1 to hNTH1 using substrate
under conditions of Vmax (400 nM
Tg:G) did not result in increased product formation (data not shown).
Interestingly, hNTH1 activity against Tg:G was stimulated by addition
of YB-1, but there was no additive effect with APE1 (data not shown).
APE1 Addition Decreases the Half-life of Covalent
Enzyme-Substrate Complexes Formed by hNTH1 with Its
Substrate--
To investigate the effect of APE1 on the reaction
intermediates of hNTH1 with its substrate, we used the reducing agent
NaBH4, to trap the covalent enzyme-substrate intermediate
(Fig. 8). NaBH4 is a strong
reducing agent, capable of reducing the Schiff base as well as existing
AP sites (26). Therefore, the trapping of the covalent reaction
intermediate with NaBH4 (Fig. 8) revealed the number of
covalent complexes present in the reaction mixture at a given time
point, in this case, 15 min. For the Tg:A substrate under steady-state
conditions, addition of APE1 resulted in a decrease in the steady-state
number of complexes initiated by hNTH1 15 min after the start of the
reaction when the rate of product formation was still linear with time.
There was no significant change in the number of steady-state complexes
with the Tg:G substrate upon APE1 addition. Addition of YB-1 resulted
in a greater number of complexes with both substrates, corroborating
our previous data (Ref. 1 and data not shown). The marked reduction in
the number of enzyme-substrate complexes (Tg:A) in the presence of both
YB-1 and APE1 is a reflection of the increased stimulation of hNTH1
turnover, resulting in almost total substrate processing within 15 min.
These data suggest that APE1 decreased the half-life of the
enzyme-substrate covalent intermediate, thereby stimulating dissociation of hNTH1 from the AP:A site without catalyzing
The generation of Tg in DNA occurs through oxidative attack on the
pyrimidine bases. Tg opposite adenine can be formed by direct hydroxyl
radical attack under aerobic conditions on thymine in situ
opposite its correctly paired base. As a blocker of both DNA and RNA
polymerases, Tg opposite adenine is a cytotoxic lesion (27). When Tg is
bypassed by a DNA polymerase, adenine is usually incorporated opposite
Tg (27). Because the 2'-deoxynucleotide triphosphate of Tg has been
shown to be a poor substrate for polymerase incorporation, formation of
Tg:G is unlikely to be the result of insertional mispairing (28). Thus,
the formation of Tg:G is due primarily to direct oxidative attack on
5-methylcytosine, resulting in deamination and oxidation, as we
previously demonstrated (3). This modification is likely to occur in
CpG islands, which are rich in 5-methylcytosine and play a role in the
regulation of gene expression. In contrast to Tg:A, Tg:G is potentially
mutagenic because, if unrepaired and bypassed by a DNA polymerase, the
insertion of adenine opposite Tg would lead to a CG We further investigated the mechanism of the difference in hNTH1
activities against Tg when it is paired with a different orphan base.
Although hNTH1 exhibited marked dissociation between its DNA
N-glycosylase and AP lyase activities when the substrate was
Tg:A, the enzyme demonstrated concomitant DNA N-glycosylase and AP lyase activities when the substrate was Tg:G. Although the rates
for base release by the enzyme were much greater for Tg:A than for
Tg:G, the rates for AP lyase activity initiated by hNTH1 against Tg:A
and Tg:G were nearly identical. Our data indicate that this discrepancy
was due to the presence or absence of hNTH1 turnover rather than
inherent differences in reaction rates.
Furthermore, we have reported that APE1 stimulated hNTH1 activity
against Tg:A by abrogation of the AP lyase step, presumably by
effecting the dissociation of hNTH1 from the AP site, thereby increasing the turnover of the DNA N-glycosylase activity of
hNTH1 (Fig. 9A). Thus, in the
presence of APE1, hNTH1 essentially becomes a monofunctional DNA
N-glycosylase against Tg:A, but not against Tg:G. Similar
results have been reported for hOGG1 (6, 7). Although we did not
observe stimulation of hNTH1 product formation by APE1 with Tg:G under
turnover conditions, we did observe APE1 stimulation of hNTH1-catalyzed
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-elimination reaction, which effects DNA strand cleavage 3'
to the abasic (AP) site formed as a product of the release of the base
from the 2'-deoxyribose moiety to which it was linked. The hydrolysis
of the Schiff base intermediate can occur in the absence of or
following enzyme-catalyzed
-elimination (AP lyase activity),
resulting in DNA strand cleavage. The enzymatic catalysis of
-elimination by DNA N-glycosylases/AP lyases has been
shown to be initiated via abstraction of the deoxyribose
pro-S-2'-hydrogen by a basic amino acid in the enzyme active
site. Several factors, including pH, can affect the efficiency of the
AP lyase step by affecting substrate binding and proton abstraction
(5).
-elimination catalysis) require the formation of the Schiff base intermediate and were thought to occur
concomitantly. However, data from our laboratory (1) and from other
laboratories (6-8) indicate that DNA N-glycosylase and
-elimination catalysis by mammalian members of the endonuclease III
enzyme superfamily are not concurrent under the assay conditions employed. We were the first to report that the DNA
N-glycosylase and AP lyase activities of hNTH1 are not
concurrent, i.e. that the rate of AP lyase-mediated strand
cleavage is much slower than the rate of DNA
N-glycosylase-mediated base release. These results are
similar to the non-concurrence of base release and strand cleavage
reported for the mammalian 8-oxoguanine-DNA
N-glycosylase homolog OGG1 (8). In this report,
we present data demonstrating that the dissociation of the two
activities of hNTH1 is dependent on the nature of the orphan base
opposite the Tg residue in DNA.
-elimination. This resulted in bypass of the AP lyase step and an increase in hOGG1
turnover. In this study, we report similar findings with hNTH1 and
APE1. However, stimulation by APE1 proved to be dependent on the nature
of the orphan base opposite the Tg residue in DNA.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was a gift from Dr.
Samuel H. Wilson (NIEHS), and APE1 was a gift from Dr. David M. Wilson III (University of California). Formamidopyrimidine-DNA
N-glycosylase was purchased from New England Biolabs Inc.,
and uracil-DNA N-glycosylase was purchased from Invitrogen.
-32P]ATP (PerkinElmer Life Sciences) and T4
polynucleotide kinase (Invitrogen) or at the 3'-end using
[
-32P]ddATP (Amersham Biosciences) and terminal
transferase (New England Biolabs Inc.). All labeled nucleotides were
purified by Sephadex G-25 spin column filtration (Amersham
Biosciences) and annealed to the complementary strand, which contained
an adenine or guanine base opposite the Tg or uracil residue.
-32P]ddAMP.
Reactions contained the indicated substrate and enzyme concentrations
and were incubated at 37 °C for 30 min. NaBH4 was added
to 100 mM, and the reactions were incubated at 37 °C for 20 min for reduction of the 2'-deoxyribose 5'-phosphate moiety (19).
The products were purified by Sephadex G-25 spin column filtration
prior to separation and analysis.
-32P]ddAMP and 100 nM hNTH1 with or
without 100 nM APE1 in a volume of 220 µl. Enzyme
reaction mixtures were incubated together for 2 min prior to initiation
of the assay upon addition of substrate. To measure base release,
5-µl aliquots were removed at the indicated time periods,
snap-frozen, and treated with 5 µl of 0.5 M putrescine (pH 8.0). The treated assay mixtures were then heated at 95 °C for 5 min, followed by addition of 10 µl of loading dye. To measure strand
cleavage and to determine incision products, 5-µl aliquots were
removed at the indicated time periods, snap-frozen, and treated with an
equal volume of 0.5 M NaBH4 at 37 °C for 20 min for reduction of the 2'-deoxyribose 5'-phosphate moiety. Samples
were then filtered through a Sephadex G-25 spin column, treated with an
equal volume of loading dye, and heated at 55 °C for 5 min, and
products were separated by 20% PAGE in 7 M urea and 1×
Tris borate/EDTA. All products were analyzed quantitatively via
phosphorimaging using a Molecular Imager FX system with Quantity One
software (Bio-Rad).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-elimination (1). However, when the substrate was Tg:G, there was no
evidence of enzyme dissociation from AP:G (2).
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Fig. 1.
hNTH1 activity against Tg:A-containing
versus Tg:G-containing 2'-deoxyribose oligonucleotide
duplex substrates. 20 nM hNTH1 was incubated with the
specified concentrations of 32P-5'-end-labeled
2'-deoxyribose oligonucleotide duplex substrate containing either an
adenine (circles) or a guanine (squares) opposite
the Tg moiety. Open symbols denote DNA
N-glycosylase activity determined by putrescine treatment of
reaction products. Closed symbols denote AP lyase activity.
Mean values were calculated from two independent experiments and did
not vary by >10%.
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Fig. 2.
Effect of APE1 on hNTH1 activity against a
Tg:A-containing 2'-deoxyribose oligonucleotide duplex substrate.
40 nM 32P-5'-end-labeled Tg:A-containing
2'-deoxyribose oligonucleotide duplex substrate was incubated with 5 nM hNTH1 with (squares) or without
(circles) 20 nM APE1. APE1 endonucleolytic
activity was abrogated in the absence of MgCl2 and the
presence of 5 mM EDTA. Open symbols denote DNA
N-glycosylase activity determined by putrescine treatment of
reaction products. Closed symbols denote AP lyase
activity.
-elimination AP
lyase products (19). The AP lyase product (p-17-p*ddA) can be seen in lanes containing hNTH1 with either the Tg:A or Tg:G substrate (lanes 2 and 4, respectively). Addition
of APE1 to hNTH1 using Tg:A produced a higher molecular weight incision
product, which was the endonucleolytic product
(p-rAP-p-17-p*ddA, where rAP is reduced
apurinic/apyrimidinic) generated by APE1 (lane 3). In the
case of the Tg:G substrate, no change in the size of the incision
product was detected upon APE1 addition, indicating that the AP lyase
activity of hNTH1 was unaffected by APE1 with this substrate
(lane 5). In Fig. 3, there are two marker lanes for the
-elimination product, one with the Tg:G-containing substrate and one
with the U:A-containing substrate (lanes 6 and 8,
respectively). Formamidopyrimidine-DNA N-glycosylase has
/
-elimination activity. Because the substrate was 3'-labeled, the
only product observed with formamidopyrimidine-DNA
N-glycosylase was the
-elimination product (lane
6). A second marker lane for the
-elimination product is the AP
site-containing substrate generated by incubation of the U:A-containing
substrate with uracil-DNA N-glycosylase and hNTH1 to
generate the AP lyase product (p-17-p*ddA) (lane
8). The U:A-containing substrate incubated with uracil-DNA
N-glycosylase and APE1 serves as the marker for the
endonucleolytic product (p-rAP-p-17-p*ddA) (lane
7).
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Fig. 3.
Effect of APE1 on the incision products
generated by hNTH1 in vitro against Tg:A- and
Tg:G-containing 2'-deoxyribose oligonucleotide duplex substrates.
Reactions contained 10 nM Tg:A- or Tg:G-containing
2'-deoxyribose oligonucleotide duplex substrate labeled at the 3'-end
with [32P]ddAMP, 20 nM hNTH1, and 10 nM APE1 as indicated. Lane 6 contained 1 unit of
formamidopyrimidine-DNA N-glycosylase (FPG).
Another marker lane for the -elimination product is also shown with
a uracil-containing radiolabeled substrate incubated with uracil-DNA
N-glycosylase (UDG) and hNTH1
(p-17-p*ddA, where the asterisk denotes the
labeled phosphate). The uracil-containing substrate was incubated with
uracil-DNA N-glycosylase and APE1 to generate a marker for
the endonucleolytic product (p-rAP-p-17-p*ddA). Reactions
were incubated at 37 °C for 30 min prior to addition of
NaBH4, followed by analysis as described under
"Experimental Procedures."
Differ in Vitro--
Because our
previous work identified YB-1 as a modifier of hNTH1 activity
(1), we next investigated whether addition of YB-1 together with
APE1 had any effect on the nature of the incision products of hNTH1
with Tg:A-containing 2'-deoxyribose oligonucleotide duplex substrate
labeled at the 3'-end with [32P]ddAMP (Fig.
4). Addition of YB-1 in the presence or
absence of APE1 increased product formation (lanes 2 and
4). However, it did not change the nature of the incision
product, which, in the presence of APE1, was the endonucleolytic
product (p-rAP-p-17-p*ddA) (lanes 3 and
4).
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Fig. 4.
Effects of APE1, YB-1, and DNA
polymerase on the incision products generated
by hNTH1 in vitro against Tg:A-containing
2'-deoxyribose oligonucleotide substrates. Reactions contained 20 mM dNTPs, 16.6 nM Tg:A-containing
2'-deoxyribose oligonucleotide duplex substrate labeled at the 3'-end
with [32P]ddAMP, 16.6 nM hNTH1, 8.3 nM APE1, 8.3 nM DNA polymerase
(
-pol), and 83 nM YB-1 as indicated.
Lane 9 contained 1 unit of formamidopyrimidine-DNA
N-glycosylase (Fpg) as a marker for the
-elimination product (p-17-p*ddA, where the
asterisk denotes the labeled phosphate). Reactions were
incubated at 37 °C for 30 min prior to addition of
NaBH4, followed by analysis as described under
"Experimental Procedures."
. Fig. 4 demonstrates the reappearance of the
-elimination
product upon addition of DNA polymerase
to hNTH1 and APE1 with a
Tg:A substrate (lane 7). This observation is consistent with
the known 2'-deoxyribose 5'-phosphatase activity of DNA polymerase
(22). The marker lane for the
-elimination product
(p-17-p*ddA) contains formamidopyrimidine-DNA
N-glycosylase (lane 9). Similar experiments with an N-terminal deletion mutant of hNTH1 lacking amino
acids 1-57 produced the same results in the presence of APE1 and YB-1
(data not shown), suggesting that the effects of YB-1 and APE1 are not
mediated by the N terminus of hNTH1.
[S] using
substrates labeled at the 3'-end with [32P]ddAMP to
determine the nature of the incision product. Because all substrate
molecules are bound by enzyme under single-turnover conditions, the
rates that determine product formation are dependent only on the rate
of base release (Schiff base formation) and
-elimination (24). If
APE1 did not affect either of these catalytic properties, then there
should be no stimulation under conditions of single turnover.
View larger version (19K):
[in a new window]
Fig. 5.
Representative plots of the effect of APE1 on
the single-turnover kinetics of hNTH1 with Tg:A-containing
(A) and Tg:G-containing (B)
2'-deoxyribose oligonucleotide duplex substrates. Reactions
contained 10 nM Tg:A-containing (A) or
Tg:G-containing (B) 2'-deoxyribose oligonucleotide duplex
substrate labeled at the 3'-end with [32P]ddAMP and 100 nM hNTH1 with (squares) or without
(circles) 100 nM APE1. Open symbols
denote DNA N-glycosylase activity determined by putrescine
treatment of reaction products. Closed symbols denote AP
lyase activity. Reactions were incubated at 37 °C for the indicated
time periods, after which aliquots were stopped by snap-freezing.
One set was treated with putrescine and the other with
NaBH4. Analysis was as described under "Experimental
Procedures."
-elimination-catalyzed cleavage of
the resulting AP site was clearly the rate-limiting reaction. Under
single-turnover conditions, the lag of the AP lyase step behind the DNA
N-glycosylase step was only slightly longer for Tg:A than
for Tg:G, indicating that the difference in processing of the two
substrates was due to enzyme turnover determined by the relative rates
of dissociation of hNTH1 from its products (Fig. 5). Further evidence
for this is shown in Fig. 6, in which
both hNTH1 and Tg:A and Tg:G substrates were present at low equimolar
concentrations. Under these conditions, the dissociation of the DNA
N-glycosylase and AP lyase activities of hNTH1 was apparent
against the Tg:A substrate, but there was no dissociation between the
two activities against the Tg:G substrate.
View larger version (15K):
[in a new window]
Fig. 6.
Representative plot of product formation by
hNTH1 against Tg:A- and Tg:G-containing 2'-deoxyribose oligonucleotide
duplex substrates as a function of time under multiple-turnover
conditions. Reactions contained 20 nM
32P-5'-end-labeled Tg:A-containing (circles) or
Tg:G-containing (squares) 2'-deoxyribose oligonucleotide
duplex substrate as indicated with 20 nM hNTH1. Open
symbols denote DNA N-glycosylase activity determined by
putrescine treatment of reaction products. Closed symbols
denote AP lyase activity.
-elimination. For all four sets of experiments, DNA
N-glycosylase activity was quantified by measuring the
cleavage of hNTH1-generated AP sites by treatment with organic base
(putrescine), which effects cleavage via
-elimination (25). In
reactions containing APE1, all cleavages were the result of APE1
activity. Addition of APE1 resulted in a 2-fold increase in the number
of hNTH1-generated AP sites compared with hNTH1 alone. Addition of YB-1
also caused a 2-3-fold increase in DNA N-glycosylase
activity, as we previously described (1). Like YB-1, APE1 increased the
kcat of hNTH1 (as a DNA
N-glycosylase) under conditions of substrate excess. The
last experiment of this group indicates that the effects of APE1 and
YB-1 were additive, resulting in a close to 4-fold increase in product.
This combined stimulation could actually be much greater because, under
our experimental conditions, most of the substrate had been processed
within 15 min.
View larger version (18K):
[in a new window]
Fig. 7.
Effect of APE1 on hNTH1 activity against a
Tg:A-containing 2'-deoxyribose oligonucleotide duplex under conditions
of Vmax. Reactions consisted of 20 nM hNTH1 incubated with 400 nM
32P-5'-end-labeled Tg:A-containing 2'-deoxyribose
oligonucleotide duplex substrate with 100 nM YB-1 and/or
100 nM APE1 as indicated for a reaction time of 15 min.
Black bars denote DNA cleavage either by hNTH1-catalyzed AP
lyase activity (in the absence of APE1) or APE1 endonucleolytic
activity (in the presence of APE1). Gray bars represent
reaction products treated with putrescine. In the absence of APE1,
these denote hNTH1 DNA N-glycosylase activity. In the
presence of APE1, all AP sites generated by hNTH1 DNA
N-glycosylase activity were endonucleolytically cleaved by
APE1. Means ± S.D. were calculated from three independent
experiments.
-elimination, consistent with results of other laboratories studying
the effect of APE1 on hOGG1 (6, 7).
View larger version (20K):
[in a new window]
Fig. 8.
Representative plot of the effect of APE1 and
YB-1 on steady-state levels of the hNTH1 Schiff base intermediate with
a Tg-containing 2'-deoxyribose oligonucleotide duplex. The
covalent enzyme-substrate intermediate was trapped using
NaBH4. Reactions contained 400 nM
32P-5'-end-labeled Tg:A-containing (black bars)
or Tg:G-containing (striped bars) 2'-deoxyribose
oligonucleotide duplex substrate with 20 nM hNTH1, 100 nM YB-1, and/or 100 nM APE1 as indicated.
Reactions were incubated at 37 °C for 15 min; NaBH4 was
then added to a concentration of 100 mM, and the reactions
were incubated for another 2 min at 37 °C prior to addition of
Laemmli SDS sample buffer.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
TA transition
mutation as well as a disruption in methylation patterns (3).
-elimination with Tg:G under single-turnover conditions (Fig.
5B). Taken together, these data suggest that hNTH1 remains
bound to the nicked AP site product. This indicates that APE1 did not
mitigate the inhibition of hNTH1 by the nicked DNA product of
-elimination of the AP:G site (Fig. 9B). Although we had
previously suggested that Schiff base hydrolysis
(k4) and product release
(k5) are not rate-limiting (1), our current data
suggest the opposite. The observation that YB-1 did stimulate Tg:G
substrate processing by hNTH1 suggests that YB-1 may effect product
release as well as the steady-state equilibrium between the Schiff base
enzyme-substrate intermediate and the noncovalent enzyme-substrate
intermediate.
View larger version (15K):
[in a new window]
Fig. 9.
Kinetic scheme for the effect of APE1 on
hNTH1 activity against Tg:A-containing (A) and
Tg:G-containing (B) 2'-deoxyribose oligonucleotide
duplex substrates. S1 is the substrate with modified
base (i.e. Tg) intact, and S2 is the substrate
containing an AP site after base release. P1 is the 3'-end
of the -eliminated 2'-deoxyribose oligonucleotide, and
P2 is the 5'-end of the
-eliminated 2'-deoxyribose
oligonucleotide. The double bond indicates the Schiff base
between the enzyme and substrate. k2 is the rate
of base release. k3 is the rate of
-elimination. k4 is the rate of Schiff base
hydrolysis. k
6 and k6
are the steady state of Schiff base resolution.
k
7 and k7 are the
binding and release of the noncovalently bound AP site moiety,
respectively. A, the inhibition of k3
by APE1 drives the reaction toward
k6/k7, the dissociation
of hNTH1 from the AP:A substrate, resulting in increased turnover, a
faster reaction rate, and a shorter half-life for the covalent
enzyme-substrate intermediate. This leaves an AP site ready for
processing by APE1 and repair synthesis. B, the effect of
APE1 on k3 results in an increased rate of AP
lyase activity. However, there is no evidence that hNTH1 is driven to
dissociate from the
-elimination product, and turnover is not
significantly affected.
What is a possible mechanistic reason for the differential activity of
hNTH1 acting on Tg:A versus Tg:G? A detailed in
vitro analysis of hNTH1 substrate processing by Eide et
al. (29) revealed that the -elimination activity of hNTH1 is
strongly dependent upon the nature of the orphan base pairing with an
AP site, being 100-fold greater with AP:G compared with AP:A. Thus, the
difference in hNTH1 processing of Tg:A versus Tg:G may be a
reflection of the affinity of hNTH1 for AP:G versus AP:A.
From our turnover data (Figs. 1 and 6), we conclude that the AP lyase
activity of hNTH1 against the AP site-containing substrate is dependent
on the binding affinity of hNTH1 for the AP site. Thus, the
dissociation of hNTH1 from the AP:A substrate following the enzymatic
release of Tg suggests a low binding affinity of hNTH1 for the AP:A
site. This is shown in the kinetic mechanism outlined in Fig.
9A, where k6 represents the
hydrolysis of the Schiff base without catalysis of
-elimination and
k7 represents the dissociation of the enzyme from the AP site. Our data suggest that APE1 drives the steady-state equilibrium from the covalent (Schiff base,
E=S2) to the noncovalent (ES2) intermediate between hNTH1 and its AP:A
substrate, resulting in the dissociation (E + S2) of hNTH1 from the AP:A site. The higher affinity of
hNTH1 for AP:G is depicted by virtually unobservable values for
k6 and k7,
in which case, all Schiff base formation results
in catalysis of
-elimination and formation of AP lyase product
(E=P2) (Fig. 9B). Because APE1
stimulates hNTH1 AP lyase activity with Tg:G without stimulating
turnover, we suggest that hNTH1 remains tightly bound to its AP lyase
product (E=P2).
The different activities that we observed with hNTH1 in vitro may be indicative of the involvement of different substrate-dependent interacting and modifying proteins in vivo. Certain lesions, by virtue of their mutagenic or toxic characteristics, may be preferentially channeled down a particular BER pathway, i.e. long- or short-patch BER. It has been suggested that the intrinsic properties of the DNA N-glycosylase involved in the initial recognition and removal of the damaged base may be responsible for BER pathway selection (30). In this case, the intrinsic properties may include the pairing base discrimination exhibited by hNTH1.
The in vivo significance of AP lyase activity is poorly
understood. The removal of the damaged base by the DNA
N-glycosylase generates an AP site, which is a labile and
therefore dangerous intermediate for the cell. AP sites are the
universal intermediates for all BER, and it is postulated that most of
these sites are processed by APE1 (31). Our studies with hNTH1 are
consistent with studies performed with mammalian OGG1, which suggested
that AP site processing is the rate-limiting step in BER initiated by
bifunctional DNA N-glycosylases/AP lyases (8). As we
previously proposed, severe oxidative stress or UV irradiation would
result in the formation of a large number of damaged bases throughout cellular DNA (1). In theory, the spontaneous removal of the damage via
concomitant DNA N-glycosylase/AP lyase activity by bifunctional DNA N-glycosylases/AP lyases would result in
the simultaneous formation of a large number of DNA strand breaks, a
cytotoxic intermediate. In support of this hypothesis, recent data on
H2O2 sensitivity in E. coli
identified the intermediates of Tg repair, rather than the persistence
of Tg, as significant contributors to cell death (32). The delayed AP
lyase activity and product inhibition inherent in hNTH1 activity may
allow for the regulation of AP site processing. The substrate-specific
product inhibition exhibited by hNTH1 may also represent the channeling of AP site processing into specific BER pathways in different regions
of the genome. The rate of removal of Tg from cellular DNA has been
shown to be very slow compared with the removal of uracil or the repair
of AP sites (22, 33). Repair of Tg has been reported to consist
mostly of short-patch BER involving DNA polymerase (33, 34). Our
data suggest that it is the AP lyase activity of DNA polymerase
that is responsible for the removal of the 2'-deoxyribose 5'-phosphate
residue left by hNTH1-catalyzed base release, followed by
APE1-catalyzed hydrolysis of the 5'-phosphate bond. The fact that DNA
polymerase
is the primary polymerase involved in the major Tg
removal pathway supports this model (34, 35). The pivotal role played
by APE1 physically connects the activities of the initial BER factors
to the DNA repair synthesis enzymes, such as DNA polymerase
, FEN1
(Flap endonuclease-1), and DNA
ligase (13, 21, 31). Data from our laboratory (1) and others
(6, 7, 36-39) have shown that interactions between BER enzymes and
other proteins can stimulate damage recognition or excision, suggesting
the probability of multiprotein complex formation in the initial events
of BER.
APE1 is not limited to its role in BER, but is a composite of the different activities of the enzyme as a redox factor (40). Notably, APE1 has been shown to control the activity of p53 through redox alteration; and in turn, p53 has been shown to play a role in BER (40-44). Like YB-1, APE1 has been shown to be a pluripotent factor and may be a key effector of the relationships between mammalian BER, oxidative signaling, transcription regulation, and cell cycle control (40, 45).
We previously demonstrated that YB-1 affects hNTH1 activity via the steady-state equilibrium between the covalent (E = S2) and noncovalent (ES2) enzyme-DNA intermediate (1). We proposed that this equilibrium may be a checkpoint for modulation of hNTH1 activity (1). In this study, we have shown that APE1 modulated hNTH1 activity at the same equilibrium point, albeit to a different effect. Thus, although YB-1 and APE1 seem to modulate hNTH1 activity in opposite ways, they have a synergistic effect in vitro. The overall effect of these two factors on hNTH1 activity is to increase hNTH1 turnover and substrate processing. Whether this has significance in vivo remains to be determined.
In conclusion, we have performed further analyses into the mechanism of hNTH1 activity and identified substrate-specific enzyme properties. We have also demonstrated a functional interaction between hNTH1 and the major mammalian AP endonuclease, APE1. This interaction is dependent on the nature of the orphan base opposite Tg.
We recently reported (2), as has another laboratory (46), that
NTH1/
mice are viable and exhibit no abnormal
phenotype. Extracts of tissues of NTH1
/
mice express an
enzyme activity that cleaves Tg:G substrates more rapidly than Tg:A
substrates. This pattern seems to be in direct contrast to the
substrate selectivity of hNTH1. Whether the substrate selectivity of
NTH1 is a reflection of actual differences in NTH1-mediated in
vivo processing of Tg:A versus Tg:G and whether the
selective stimulatory effects of APE1 affect processing await further elucidation.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. David M. Wilson III for
supplying APE1 and Dr. Samuel H. Wilson for supplying DNA polymerase
.
![]() |
FOOTNOTES |
---|
* This work was supported by the Department of Pathology, New York University School of Medicine, and an award from the New York University School of Medicine Research Bridging Support Fund (to G. W. T.) and by NIEHS Grant ES 09127 (to A. K. B.) and NIH Grant GM 46312 (to R. P. C.) from the National Institutes of Health.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
Pathology, New York University Medical Center, 550 First Ave., New
York, NY 10016. Tel.: 212-263-5473; Fax: 212-263-8211; E-mail:
george.teebor@med.nyu.edu.
Published, JBC Papers in Press, January 8, 2003, DOI 10.1074/jbc.M212168200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: hNTH1, human NTH1; AP, apurinic/apyrimidinic; BER, base excision repair; Tg, thymine glycol; hOGG1, human OGG1.
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