(Received for publication, April 27, 1996, and in revised form, September 4, 1996)
From the Sealy Center for Molecular Science,
University of Texas Medical Branch, Galveston, Texas 77555 and
¶ Ares Inc., Randolph, Massachusetts 02368
Apurinic/apyrimidinic endonuclease (AP endo)
makes a single nick 5 to a DNA abasic site. We have characterized this
reaction by steady-state and transient-state kinetics with purified
human AP endo, which had been expressed in Escherichia
coli. The substrate was a 49-base pair oligonucleotide with an
abasic site at position 21. This substrate was generated by treating a
49-mer duplex oligonucleotide with a single G/U located at position 21 with uracil-DNA glycosylase. The enzymatic products of the AP endo
nicking reaction were a 20-mer with a hydroxyl group at the 3
-terminus
and a 28-mer with a phosphodeoxyribose at the 5
-terminus. To obtain
maximal enzymatic activity, it was necessary to stabilize the abasic
site during treatment with uracil-DNA glycosylase with a reducing
agent. Otherwise, a 20-mer with phosphoribose at the 3
-terminus
resulted from
-elimination. In agreement with others,
Km and kcat were 100 nM and 10 s
1, respectively. Heat treatment of
the abasic site-containing 49-mer without enzyme also resulted in
conversion to the
-elimination product. The resultant heat
degradation product was an efficient inhibitor of AP endo with a
Ki of 30 nM. The enzyme required divalent cation (Mg2+) for activity, but bound substrate
DNA in the absence of Mg2+. Electrophoretic mobility shift
assays indicated that AP endo bound tightly to DNA containing an abasic
site and formed a 1:1 complex at low enzyme concentrations. The
association and dissociation rate constants for substrate binding to AP
endo were determined by using a challenge assay to follow AP
endo-substrate complex formation. Heat degradation product together
with heparin served as an effective trap for free enzyme. The results
are consistent with a Briggs-Haldane mechanism where
kon and koff are 5 × 107 M
1 s
1 and
0.04 s
1, respectively (Kd = 0.8 nM), kcat is 10 s
1,
and product release is very rapid (i.e.
koff,product
10 s
1). This scheme
is in excellent agreement with the measured steady-state kinetic
parameters.
Human apurinic/apyrimidinic endonuclease (AP
endo)1 (EC 3.1.25.2) is a bifunctional
enzyme with the ability both to initiate repair of abasic sites in
damaged DNA and to act as a redox protein in restoring promoter binding
to oxidation-damaged Jun-Fos or Jun-Jun dimers (1, 2). The gene for
human AP endo has been identified (3, 4, 5, 6), and several recombinant AP endos have been expressed (4, 5, 6). The open reading frame encodes a
protein of 319 amino acids and a molecular mass of 35.5 kDa. In this
report, we focus on the endonuclease function that is found in the
carboxyl-terminal 250 residues (2, 7). AP endo makes a single cleavage
in the phosphodiester backbone on the 5-side of the abasic site
created by any of several DNA glycosylases and is, therefore,
classified as a class II endonuclease (Fig. 1) (8, 9, 10). Subsequently,
in mammalian base excision repair, the phosphodeoxyribose remaining
attached to the 5
-end of the downstream DNA strand is removed by the
amino-terminal 8-kDa domain of DNA polymerase
(11) in order for
this polymerase to insert a nucleotide opposite the exposed base on the
complementary strand (12, 13).
The endonuclease activity of AP endo has been well characterized for its preference for abasic site-containing DNA and by steady-state kinetic analysis in the presence of divalent cation. In order to understand the kinetic basis for enzyme specificity and efficiency, each step in the reaction pathway needs to be characterized thermodynamically and kinetically. This is a prerequisite to rational drug design in developing efficient enzyme inhibitors to down-regulate DNA repair. In this paper, we examine substrate binding in detail to derive association and dissociation rate and equilibrium constants. These studies were facilitated by the observation that the spontaneous heat degradation product (HDP) of the oligonucleotide containing an abasic site is an effective inhibitor of human AP endo. The HDP was employed in a challenge assay to measure enzyme-DNA complex formation. Product complexes could not be formed during catalytic assays, indicating that product release was much faster than the nicking reaction. These results suggest a simple Briggs-Haldane mechanism for AP endo.
The substrate for all enzyme assays, obtained either from Operon Technologies, Inc. (Alameda, CA) or from Genosys (Woodlands, TX), was a 49-base pair (bp) oligomer containing a single G/U base pair at position 21.
![]() |
![]() |
![]() |
The single-stranded oligomer was labeled at the 5-end of the
U-containing strand by means of T4 polynucleotide kinase (New England
Biolabs) and [
-32P]ATP (Amersham Corp.) in the buffer
supplied by the manufacturer. End-labeling was performed at 37 °C
for 45 min. In some cases, the 49-mer was labeled at the 3
-end by
means of terminal deoxynucleotidyl transferase (Promega Corp.) and
[
-32P]ddATP (Amersham Corp.). The abasic site was
created by treating the double-stranded oligomer with uracil-DNA
glycosylase (Epicentre Technologies) at 1 unit of enzyme/100 pmol of
uracil residues, for 15-30 min at 37 °C. DNA substrate was
quantified spectrophometrically. The buffer consisted of 50 mM HEPES-NaOH, pH 7.5, 0.1 mM EDTA, and 0.1 mM freshly prepared NaBH4, as indicated. The
reaction was terminated by heating at 70 °C for 5 min. The mixture
was allowed to cool slowly to room temperature.
Steady-state enzyme assays were carried out at 25 °C for 5-120 s in 50 mM HEPES-NaOH, pH 7.5, 25 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA, 0.02-20 nM AP endo, and 150 nM DNA in a volume of 5 µl. Assays were terminated either by the addition of urea-containing gel loading dyes or with EDTA to a final concentration of 70-85 mM. Products were resolved by gel electrophoresis using 8 M urea, 15% polyacrylamide gels (14), followed by autoradiography, and quantified with a Molecular Dynamics PhosphorImager (Sunnyvale, CA). In some cases, products were restabilized before electrophoresis by treatment with 0.1 M NaBH4 for 30 min at 0 °C, followed by ethanol precipitation in the presence of 0.2 µg/ml tRNA. Enzyme activity was linear for up to 60 s in the range of 0.02-10 nM AP endo.
Challenge AssayAP endo could be limited to a single turnover by the addition of an enzyme trapping agent. Enzyme was preincubated with substrate, but without Mg2+, to form enzyme-substrate complex in the presence of 4 mM EDTA. Productive complex was then assayed by the addition of excess Mg2+ and trap (i.e. heparin and HDP). Heparin and HDP bind to free enzyme, as well as enzyme that dissociates from the substrate during the course of the reaction, making the dissociation reaction irreversible.
Transient-state assays to determine the association and dissociation rate constants were carried out under single-turnover conditions. To determine the association and dissociation rate constants, kon and koff, respectively, enzyme and substrate were incubated for various periods up to 30 s under pseudo-first order conditions (i.e. substrate/enzyme = 10). At the end of each period, the amount of enzyme-DNA complex was measured by initiating enzymatic activity with the addition of Mg2+ (10 mM final concentration) and trap and incubating for an additional 10 s. The final volume was 5 µl. The reaction was terminated by the addition of EDTA to a final concentration of 87 mM. Further details are outlined in the figure legend.
Electrophoretic Mobility Shift AssaySubstrate DNA was prepared as described above, except that NaBH4 was omitted during generation of the abasic site. To prevent the formation of HDP, the reaction was not heated to terminate the uracil-DNA glycosylase step. Substrate DNA was incubated with AP endo at different concentrations for 45 min at 25 °C in the presence of 0.5% polyvinyl alcohol, 10% glycerol, 100 µg/ml bovine serum albumin, 8 mM NaCl, 50 mM HEPES-NaOH, pH 7.5, 0.1 mM EDTA, and 5 mM MgCl2. The inclusion of MgCl2 was not necessary for binding. The incubation mixture was loaded onto a 7% nondenaturing polyacrylamide gel, and components were resolved by electrophoresis at 90 V (7 V/cm) for 6 h. The distribution of oligodeoxynucleotide bound and unbound forms was quantified by PhosphorImager analysis.
Data AnalysisData were fitted to appropriate equations by nonlinear least squares methods. The association rate constant and steady-state time courses were simulated with HopKINSIM (15), a Macintosh version of the kinetic simulation program KINSIM (16).
The association and dissociation rate constants were determined from
the binding curves determined under pseudo-first order conditions.
Assuming a simple one-step binding model (E + S ES), the data should conform to a simple exponential where
kobs = kon[S] + koff. The association and dissociation rate
constants were determined from a secondary plot of kobs
versus [S].
The HAP1h cDNA (3) was
amplified by polymerase chain reaction from HeLa cell DNA using the
following primers:
5-AAAA
ATGCCGAAACGTGG-3
(BAP.NsiI) and
5
-CTC
TCACAGAGCTAGGTATAGGGTAA-3
(BAP.PacI).
The BAP.NsiI primer added an NsiI restriction site
(underlined) and created an NdeI restriction site
(italicized) upstream of the coding sequence, which is shown in bold.
The BAP.PacI primer added a PacI restriction site
(underlined) downstream of the coding sequence. The resulting
polymerase chain reaction product was digested with NsiI and
PacI and inserted into plasmid vector pXC20PacI, which had
been digested with the same two enzymes. Vector pXC20PacI is a
derivative of the phage PL promotor vector
pXC36 (17) and will be described elsewhere. The resulting plasmid was
named pXC49, and the nucleotide sequence of the insert was confirmed by
DNA sequencing. A restriction fragment containing the entire HAP1h
coding sequence plus a short segment of the vector 3
to the coding
sequence was prepared by complete digestion of pXC49 with
BamHI followed by partial digestion with NdeI.
The fragment was inserted into the phage T7 promotor expression vector
pET3a (18), which had been digested to completion with NdeI
and BamHI. The HAP1h expression vector this created was
named pXC53. The structure of pXC53 was confirmed by restriction and
DNA sequence analysis. Plasmids pXC49 and pXC53 were constructed as
part of a larger study of HAP1h expression in Escherichia
coli and will be described in more detail elsewhere.
Human AP endo was
expressed in E. coli strain BL21/DE3pLysS from pXC53
carrying the HAP1h gene. After induction with
isopropyl-1-thio--D-galactopyranoside for 2 h,
cells from a 2-liter culture were lysed in Buffer A (50 mM
Tris-HCl, pH 7.5, 1 mM EDTA) containing 500 mM
NaCl, 0.5 mM phenylmethylsulfonyl fluoride, 1.0 µg/ml
pepstatin, and 1 mM dithiothreitol by means of sonic
disruption at 0-1 °C. After removal of cell debris by
centrifugation at 27,000 × g for 15 min, the
supernatant fraction was adjusted to 100 mM NaCl by
addition of Buffer A containing protease inhibitors and dithiothreitol.
This was passed over a Q-Sepharose column (100 ml bed volume) connected
in series to an S-Sepharose column of the same size. After washing with
Buffer A containing 100 mM NaCl, the S-Sepharose column was
developed with increasing concentrations of NaCl up to 1 M.
AP endo, eluting at high salt (>500 mM NaCl), was further
purified by chromatography over a Mono S column by FPLC (Pharmacia
Biotech Inc.). The purified enzyme, containing no measurable nuclease
activity with double-stranded DNA substrate, eluted from the Mono S
column at approximately 450 mM NaCl. The entire
purification could be achieved in 2 days with a yield of greater than 6 mg of pure enzyme/liter of E. coli culture. Enzyme
concentration was determined from Bradford assays (19), which had been
calibrated by amino acid analysis. The ratio of protein concentration
determined by amino acid analysis to that determined by the Bradford
assay was 0.37. Amino-terminal sequencing of the purified enzyme
indicated a single protein beginning with Pro2 of the human
AP endo open reading frame. Truncation of the natural Met1
has also been found for many other E. coli recombinant DNA
enzymes.
The AP site in DNA exists as an
equilibrium mixture of the open-chain aldehyde, the - and
-hemiacetals, and the open-chain hydrate. Although open-chain
aldehydes constitute a very small percentage of total AP sites, they
are subject to
-elimination at higher temperatures and in the
presence of a variety of agents including common buffers (8, 9, 20)
(Fig. 1, form III). The
-elimination
product itself can be further shortened to the
,
-elimination
product if strong base is also present during heat exposure (Fig. 1,
form II).
-Elimination is prevented, however, by
NaBH4 reduction of the open aldehyde to deoxyribitol.
Beginning with a 49-bp oligodeoxynucleotide substrate, we examined
various potential phosphodiester backbone cleavage products. Fig.
2A illustrates the difference in
electrophoretic mobility of the form III
-elimination cleavage
product created by treating the unstabilized 49-bp abasic site-containing substrate molecule at 70 °C (lane 10),
the form II product created by treatment with both heat and base
(lane 9), and the enzymatically generated form I 20-mer with
a free 3
-hydroxyl group at its terminus (lanes 6-8). Heat
treatment of the stabilized substrate did not alter the substrate
(lane 12), while treatment of the stabilized 49-bp abasic
site-containing substrate with both heat and strong base resulted in a
product with mobility of form I 20-mer containing the 3
-hydroxyl group at its terminus (lane 11). This latter product has
been proposed to involve a 3
,5
cyclization intermediate (21).
Comparison of human AP endo activity with substrates generated in the
presence or absence of NaBH4 is also shown in Fig. 2 (lanes 1-8). The rate of the cleavage reaction was faster
with the stabilized substrate, and products other than the anticipated form I 20-mer were not detected (Fig. 2). While the absence of undesired spontaneous -elimination products was not a surprise for
the NaBH4-treated substrate, an enhancement of the
catalytic rate was unexpected.
With the stabilized
substrate, formation of form I product was linear for 60 s at an
enzyme concentration of 0.02 nM. The apparent
Km and kcat were 100 nM and 10 s1, respectively (Fig.
3A and Table I). Enzyme
activity required divalent cation (Mg2+) and was completely
blocked by 4 mM EDTA in the absence of added Mg2+.
|
To understand the observed rate enhancement with the stabilized
substrate, the spontaneous -elimination product (Fig. 1, form
III) was deliberately generated by heating unstabilized substrate without enzyme for 30 min at 70 °C. When this HDP was included in
the reaction mix, it proved to be an inhibitor of AP endo activity (Fig. 3). When inhibition was studied as a function of stabilized substrate concentration, HDP increased the apparent
Km for stabilized substrate without markedly
altering Vmax (i.e. ordinate-intercept; Fig. 3A). From Dixon analysis of the
inhibition, the Ki for this competitive inhibitor
was 30 nM (Fig. 3B). Thus the enhancement of AP
endo activity for reactions in which the abasic site DNA substrate was
stabilized by NaBH4 reduction was probably due to the
absence of spontaneously generated HDP, which could have accumulated in
the reaction mixture. This proposal is consistent with the degree of
inhibition observed with spontaneously generated HDP (80%, Fig.
2B) and that determined here with deliberately generated HDP
(Fig. 3), where inhibition under the conditions described in Fig. 2
would be expected to be 60%.
To assign the stoichiometry of binding of
AP endo and DNA, we used an electrophoretic mobility shift assay to
measure the relative amount of enzyme-DNA complex formed at various
enzyme/DNA ratios (Fig. 4). There is more complex formed
with an abasic site-containing oligomer than with a double-stranded
oligomer without an abasic site, indicating that binding to substrate
or nicked DNA was much tighter than binding to "intact"
double-stranded DNA. The apparent equilibrium dissociation constant for
the "abasic site" DNA was less than 0.1 nM. The enzyme
concentration saturating the abasic site-containing DNA corresponded to
a molecular ratio of 1 enzyme to 1 DNA molecule (Fig.
4B).
To delineate the individual components of substrate DNA binding that are embedded within the Michaelis constant (Km), we measured substrate association and dissociation with AP endo by using a challenger to trap free enzyme, in order to estimate the concentration of enzyme-substrate complex. In the presence of challenger, enzyme that is bound productively to substrate can nick DNA, whereas free enzyme is quenched. Thus, activity is proportional to the concentration of ES complex. Various agents including single-stranded DNA, oligo(dT), higher concentrations of unlabeled abasic site-containing substrate, heparin, and HDP were evaluated for their ability to trap free enzyme and prevent catalytic cycling. An effective trap should quench catalytic cycling when preincubated with enzyme and substrate. Single-stranded DNA, oligo(dT), and unlabeled abasic site-containing substrate were ineffective traps. Unlabeled abasic site-containing substrate probably was not a good trapping agent, since it is an excellent substrate for human AP endonuclease as suggested by the high turnover number and low Km. Accordingly, attempts to use unlabeled substrate resulted in depletion of both labeled and unlabeled substrate at the concentration of enzyme used to perform single-turnover experiments. However, of particular interest were the results with HDP and heparin. HDP or heparin alone were ineffective in completely preventing cycling of the enzyme. However, in the presence of both heparin and HDP, the enzyme was prevented from catalytic cycling over a 10-s interval (data not shown). In addition, the amount of product formed per catalytic cycle was directly proportional to enzyme concentration. Thus, the combination of heparin and HDP effectively competes for substrate binding without being "consumed," making this mixture a better trap than unlabeled substrate.
The association (kon) and dissociation
(koff) rate constants for the AP endo-substrate
interaction were determined by examining the time dependence of
ES formation by employing the challenge assay as described
above. To follow ES formation, enzyme and substrate were
incubated under pseudo-first order conditions in the absence of
Mg2+ for periods up to 30 s (Fig. 5).
For each period, the amount of ES complex was measured by
initiating enzymatic activity with Mg2+ and trap. Substrate
was able to bind to AP endo in a concentration-dependent manner in the absence of Mg2+. The apparent half-time
(t1/2) for enzyme and DNA association was 7.4 and
2.9 s for 0.1 nM and 0.4 nM enzyme,
respectively, where t1/2 is ln
2/kobs. Assuming a simple binding model
(E + S ES), these half-times correspond to an
apparent association rate constant of 5 × 107
M
1 s
1 and suggest that the
dissociation rate constant is very slow (koff
0.04 s
1). The corresponding equilibrium dissociation
constant is, therefore, 0.8 nM. Longer incubations
(i.e. 5 min) with several enzyme and substrate
concentrations (1-10 nM) resulted in an equilibrium level
of complex formation consistent with this dissociation constant. This
equilibrium constant is larger than that determined by electrophoretic mobility shift assay (Fig. 3). Therefore, the equilibrium constant determined by electrophoretic mobility shift assay appeared to substantially overestimate DNA binding affinity.
Rate-limiting Step
When product release is slower than
chemistry, the time course of product formation is biphasic. There is
an initial burst of product formation equivalent to the active enzyme
fraction, followed by a linear phase that represents slow product
dissociation. However, the rapid consumption of substrate in the
presence of high enzyme concentrations precluded accurate estimates of
product formation at times accessible by manual mixing and sampling
techniques. Results of Wilson et al. (22) suggest that
chemistry of the enzymatic reaction is itself rate-limiting. In their
study, the presence of a 5-phosphorothioate derivative at the scissile
bond phosphate substantially reduced the rate of the enzymatic
reaction, an observation that is consistent with an elemental effect on the nicking reaction. These results indicate that chemical cleavage or
a conformational change prior to nicking is rate-limiting, instead of a
subsequent step such as product release. If steps occurring after
chemistry are so rapid that they are not rate-limiting, the ratio of
kcat/Km is a measure of the
apparent second order rate constant for productive substrate binding.
In keeping with this idea, it is noted that the values for
kcat/Km and the association
rate constant for AP endo are comparable (Table I). Additionally,
kinetic simulation of substrate binding from these steady-state
measurements (Fig. 5, dotted lines), assuming an
irreversible binding reaction, demonstrate that there is good agreement
with the binding time courses determined by the challenge assay (Fig.
5, solid lines). The amplitude of the simulated time courses
is reduced to that observed for the time courses of complex formation
determined with a trapping agent, if a dissociation rate constant of
0.10 s
1 is included (data not shown).
The results described above can be used to derive a minimal kinetic scheme consistent with a Briggs-Haldane mechanism (Scheme I). Additionally, steady-state kinetic parameters can be calculated or derived from simulation of the model depicted in Scheme I for comparison to observed values (Table I). In this scheme, product release (i.e. k3) is assumed to be rapid since a phosphorothioelemental effect has been reported (22), and kcat reflects the chemical step (i.e. k2).
![]() |
![]() |
In this study, we used steady-state and
transient-state kinetic parameters to describe human AP endo enzymatic
nicking of a synthetic oligomer with a single abasic site. This study
was facilitated by the observation that the spontaneous heat
degradation product of abasic site-containing DNA inhibits the enzyme
competitively. The minimal mechanism proposed is a simple
Briggs-Haldane scheme, where the forward catalytic reaction competes
with the dissociation of the enzyme-DNA complex. This kinetic mechanism
is a prerequisite for studies of the kinetic properties of mutant or
altered enzyme forms and will facilitate identification of
chemotherapeutic agents designed to inhibit DNA repair. Definitive
evidence is lacking that AP endo participates in base excision repair.
However, since the turnover number for DNA polymerase , known to be
integral in the single-nucleotide base excision repair pathway (13), is
approximately 1 s
1 (23, 24), AP endo would not be
rate-limiting if operating under optimal conditions. It remains to be
determined if human AP endo enzyme activity can be modulated in
vivo and if this will translate to altered sensitivity to DNA
damaging agents repaired by base excision repair.
We thank Dr. Veronique Bailly and Katherine Latham for helpful discussions regarding stabilization of the abasic site.