Báo cáo khoa học: Chinese hamster apurinic⁄apyrimidinic endonuclease (chAPE1) expressed in sf9 cells reveals that its endonuclease activity is regulated by phosphorylation docx

Chinese hamster apurinic
⁄
apyrimidinic endonuclease
(chAPE1) expressed in sf9 cells reveals that its
endonuclease activity is regulated by phosphorylation
Mandula Borjigin
1
, Bobbie Martinez
2
, Sarla Purohit
2
, Gaudalupe de la Rosa
2
, Pablo Arenaz
2
and
Boguslaw Stec
3
1 Department of Chemistry, Bowling Green State University, OH, USA
2 Department of Biological Sciences, Department of Chemistry, University of Texas, El Paso, USA
3 Sanford-Burnham Medical Research Institute, La Jolla, CA, USA
Introduction
The mammalian apurinic ⁄ apyrimidinic endonuclease
(APE1) is a multifunctional protein that plays an
essential role in DNA repair and gene regulation [1].
In particular, it is a critical component of the base
excision repair pathway, which is employed to repair
damaged DNA. The base excision repair pathway is
initiated by spontaneous or enzymatic N-glycosidic
bond cleavage creating an abasic site in DNA [2]. Aba-
sic sites in DNA alter genetic information and hinder

normal cellular activity, posing a major threat to the
integrity of the DNA molecule and the survival of the
cell [3–5]. The importance of APE1 is also underscored
by the fact that homozygous knockout mice are
embryonic lethal [6]. The mechanism of its prominent
Keywords
apurinic endonuclease; caseine kinase
phsphorylation; DNA repair; enzyme
kinetics; ICP; regulation by phosphorylation
Correspondence
B. Stec, Sanford-Burnham Medical Research
Institute, 10901 N. Torrey Pines Rd,
La Jolla, CA 92037, USA
Fax: 858 795 5225
Tel: 858 795 5257
E-mail:
M. Borjigin, Department of Chemistry, 144
Overman Hall, Bowling Green State
University, Bowling Green, OH 43403, USA
Fax: 419 372 8088
Tel: 419 372 8088
E-mail:
(Received 7 June 2010, revised 30 August
2010, accepted 10 September 2010)
doi:10.1111/j.1742-4658.2010.07879.x
Apurinic ⁄ apyrimidinic endonuclease (APE), an essential DNA repair
enzyme, initiates the base excision repair pathway by creating a nick 5¢ to
an abasic site in double-stranded DNA. Although the Chinese hamster
ovary cells remain an important model for DNA repair studies, the Chinese
hamster APE (chAPE1) has not been studied in vitro in respect to its

kinetic characteristics. Here we report the results of a kinetic study per-
formed on cloned and overexpressed enzyme in sf9 cells. The kinetic
parameters were fully compatible with the broad range of kinetic parame-
ters reported for the human enzyme. However, the activity measures
depended on the time point of the culture. We applied inductivity coupled
plasma spectrometry to measure the phosphorylation level of chAPE1. Our
data showed that a higher phosphorylation of chAPE1 in the expression
host was correlated to a lower endonuclease activity. The phosphorylation
of a higher activity batch of chAPE1 by casein kinase II decreased the
endonuclease activity, and the dephosphorylation of chAPE1 by lambda
phosphatase increased the endonuclease activity. The exonuclease activity
of chAPE1 was not observed in our kinetic analysis. The results suggest
that noticeable divergence in reported activity levels for the human APE1
endonuclease might be caused by unaccounted phosphorylation. Our data
also demonstrate that only selected kinases and phosphatases exert regula-
tory effects on chAPE1 endonuclease activity, suggesting further that this
regulatory mechanism may function in vivo to turn on and off the function
of this important enzyme in different organisms.
Abbreviations
APE, apurinic ⁄ apyrimidinic endonuclease; ChAPE1, Chinese hamster apurinic ⁄ apyrimidinic endonuclease; CK I, casein kinase I;
CK II, casein kinase II; ICP, inductivity coupled plasma.
4732 FEBS Journal 277 (2010) 4732–4740 ª 2010 The Authors Journal compilation ª 2010 FEBS
endonuclease activity is that the enzyme incises the
phosphodiester backbone 5¢ next to the abasic site
(cleaving P-O-3¢ bond), leaving a 3¢-OH and a 5¢ deoxy-
ribose phosphate [7]. Other important functions are
duplex-specific 3¢–5¢ exonuclease activity, 3¢-repair
phosphodiesterase activity, 3¢-phosphatase activity and
RNase H activity [8–11].
Although many mammalian APEs were studied, the

APE1 from Chinese hamster ovary cells was not stud-
ied in vitro, despite being an important model for
DNA repair mechanisms [12]. This is an important
enzyme and Chinese hamster APE (chAPE1) should
provide additional data that can bridge the gap
between mouse and human models. There are quite
noticeable discrepancies in reports concerning two
major catalytic (endonuclease and 3¢–5¢ exonuclease)
activities reported for several species. There is a sub-
stantial spread in the level of endonuclease activity
reported for human APE1, with K
m
ranges from 3.4 to
200 nm, k
cat
from 1.38 to 10 s
)1
and k
cat
⁄ K
m
from 0.05
to 0.5 nm
)1
Æs
)1
[13–18]. There is also controversy with
regard to the 3¢–5¢ exonuclease activity of human
APE1, for which robust activity has been reported
[19,20], a much lower level ( 100–10 000-fold lower)

than its endonuclease activity [13,21,22] or no measur-
able activity [23–25]. For instance, the murine APE
had approximately the same level of 3¢–5¢ exonuclease
activities as its endonuclease activity [26,27] and the
bovine and rat APE1 expressed in the bacterial cell do
not exhibit 3¢–5¢ exonuclease activity [28,29].
Here we report the results of studies performed on
the recombinant protein (chAPE1) using a steady-state
kinetics method with radiolabeled substrates and an
electrophoretic gel assay. The kinetic constants
obtained it this study fell into the expected range, tak-
ing into account the identity level compared with
human enzyme (92%) and mouse enzyme (94%).
However, we noticed significant variation from batch
to batch of the enzyme, which was reminiscent of the
abovementioned results. We hypothesize that the phos-
phorylation might be responsible for a broad range of
activity levels. We also speculate that the results
obtained for chAPE1 might have full relevance to the
results obtained for highly homologous mammalian
endonucleases.
The phosphorylation level of chAPE1 was quantita-
tively analyzed by measuring the phosphate amount of
protein using inductivity coupled plasma (ICP) spec-
trometry. The endonuclease activity rate constant of
the differentially phosphorylated naturally expressed
chAPE1 was obtained by performing a steady-state
kinetics analysis. To further verify the phosphorylation
effects on chAPE1 endonuclease activity, the protein
was subject to casein kinase I (CK I) or casein kinase

II (CK II) and dephosphorylated with lambda phos-
phatase or alkaline phosphatase. Their effects were
quantified by performing the endonuclease assay and
the kinetic parameters were obtained by fitting the
data into a Michaelis–Menten model. We did not
detect perceivable exonuclease activity of chAPE1 in
our study.
Results
Overexpression of chAPE1 in the sf9 cell line and
its purification
The expression level of chAPE1 in insect cells infected
by the recombinant baculovirus with a multiplicity of
infection of eight reached the plateau at 48 h and
declined after 72 h postinfection. The western blot
showed the expression level in the selected time course
(Fig. 1). The protein was purified using a Ni-NTA col-
umn and a size exclusion column and the histidine tag
was cleaved with enterokinase; the native protein
appeared as a band at  35.5 kDa (Fig. 2). The purity
was > 90%, as judged by gel electrophoresis.
Fig. 1. Expression profile of chAPE1 in sf9 cells. Western blot
autoradiograph: Lanes 1–8 correspond to the time points of chA-
PE1 expression after infection by recombinant virus. The time
points are 0, 6, 12, 18, 24, 48, 60 and 72 h, respectively.
Fig. 2. SDS ⁄ PAGE of chAPE1 stained with Coommassie Blue. In
the left-hand lane are protein markers; the lanes to the right are dif-
ferent concentrations of chAPE1 purified from sf9 cells by running
Ni-NTA and Sepherose 75 columns.
M. Borjigin et al. Phosphorylation controls chAPE1 activity
FEBS Journal 277 (2010) 4732–4740 ª 2010 The Authors Journal compilation ª 2010 FEBS 4733

Different endonuclease activity levels at different
time points of the expression
We carried out the endonuclease activity screen for
chAPE1 from different time points (24, 48 and 72 h)
of three different batches of sf9 cell culture. Because
of the convenience and reliability of the kinetic
parameter for either the first or pseudo-first order
reaction scheme, we measured K
obs
of the catalytic
activity, using 100 nm abasic DNA and 5 nm enzyme.
The density counts of the product and the substrate
at each time point were quantified from the gel auto-
radiograph using the Phosphoimager software, quan-
tity one. A typical gel image of the chAPE1
endonuclease catalysis is shown in Fig. 3A. The activ-
ity level also peaked at the 24 h postinfection expres-
sion time point, with a 1.8-fold higher activity than
at the 72 h time point and 1.4-fold higher than at
48 h (Fig. 3B).
Effects of phosphorylation on endonuclease
activity of chAPE1
We initiated the investigation of the phosphorylation
effects by measuring the phosphate amount of chAPE1
from the nine samples studied above. The estimated
number of phosphorylated residues in the chAPE1
sample was 9.6 (at 24 h), 15.4 (at 48 h) and 18.0 (at
72 h) (Table 1). The phosphorylation level correlated
quite well with the endonuclease activity level mea-
sured above. The higher the level of phosphorylation

of chAPE1 the lower the endonuclease activity, and
conversely the lower the phosphorylation the higher
the activity level. In order to validate this statement,
we phosphorylated the batch of chAPE1 (24 h time
point sample with the highest activity), measured its
activity level and dephosphorylated the same sample of
chAPE1 to reverse the phosphorylation effect.
The chAPE1 harvested at the 24 h time point was
phosphorylated with either CK I or CK II and
dephosphorylated with lambda phosphatase or alkaline
phosphatase to measure its endonuclease activity. CK
II decreased the rate constant ( K
obs
) of chAPE1 by
6.2-fold and lambda phosphatase elevated the activity
by 2.1-fold, whereas CK I and alkaline phosphatase
did not affect the activity level (Fig. 4). When the
A
B
Fig. 3. APE activity of chAPE1 and the initial screen at several time points. (A) APE catalytic activity was shown at the designated time
points of the reaction. The top bands are the substrate and the bottom bands are the product. (B) The batches at the 24 h time point had an
activity level 1.4-fold higher than that at 48 h and 1.8-fold higher than that at 72 h.
Table 1. The phosphorylation state of the recombinant chAPE1 at
three different time points of expression. The recombinant chAPE1
has 44 potential phosphorylation sites (serine, threonine, tyrosine).
The protein contains 11 sulfur atoms (in methionine, cystine), and
its molecular mass is 35.5 kDa. The mole number of chAPE1 was
calculated using mass divided by the molecular mass. The mole
number of sulfur in the sample was also calculated and was used
as the reference (or standard). The number of phosphorus atoms in

a chAPE1molecule was calculated by dividing the mole number of
phosphorus by the mole number of the protein. The R
2
values in
the linear regression of the standard curves were higher than
0.9998.
Time
Concentration
of chAPE1
lgÆmL
)1
Phosphorus
measured
lgÆmL
)1
Sulfur
measured
lgÆmL
)1
Number of
phosphorus
atoms in a
chAPE1
molecule
24 h
Sample 1
252.6
2.14 2.53
9.6 ± 0.2Sample 2 2.07 2.46
Sample 3 2.16 2.55

48 h
Sample 1
250.4
3.47 2.63
15.4 ± 0.4Sample 2 3.29 2.45
Sample 3 3.37 2.59
72 h
Sample 1
249.1
3.86 2.42
17.97 ± 0.2Sample 2 3.91 2.47
Sample 3 3.96 2.52
Phosphorylation controls chAPE1 activity M. Borjigin et al.
4734 FEBS Journal 277 (2010) 4732–4740 ª 2010 The Authors Journal compilation ª 2010 FEBS
chAPE1 phosphorylated by either CK I or CK II was
dephosphorylated with lambda phosphatase, the endo-
nuclease activity was restored to the highest level
(Fig. 4).
Steady-state kinetic studies of chAPE1
endonuclease activity
We carried out the endonuclease kinetic analysis on
phosphorylated or dephosphorylated chAPE1, using a
Michaelis–Menten model. The dephosphorylation of
chAPE1 by lambda phosphatase increased the endonu-
clease activity (k
cat
⁄ K
m
) by 16.7-fold relative to the
activity of chAPE1 phosphorylated by CK II. The

kinetic parameters for chAPE1 phosphorylated by CK
II were k
cat
= 0.58 ± 0.02 s
)1
, K
m
= 81 ± 10.59 nm
and k
cat
⁄ K
m
= 7.2 · 10
)3
nm
)1
Æs
)1
. The parameter
values for chAPE1 dephosphorylated by lambda
phosphatase were k
cat
= 5.67 ± 0.21 s
)1
, K
m
=48±
6.98 nm and k
cat
⁄ K

m
= 0.12 nm
)1
Æs
)1
(Fig. 5). These
results, along with the K
obs
values from the initial
screen and ICP data, show that phosphorylation
regulates chAPE1 endonuclease activity in vitro and
that chAPE1 expressed in sf9 cell has a different
level of phosphorylation at different time points of
expression.
In the same manner as the initial endonuclease
assay, exonuclease activity was tested for untreated
chAPE1, phosphorylated and dephosphorylated chA-
PE1 with a much higher enzyme concentration
(100 nm), as described in the experimental proce-
dures section. No detectable exonuclease activity
was observed up to 720 s (Fig. 6) in any of these
conditions.
Discussion
Here we studied the APE from the Chinese hamster
ovary cell, an important model for DNA repair. The
Chinese hamster and its cell lines have been the para-
digm for DNA repair research at cellular and gene reg-
ulation levels for several decades [30–36] and its APE
(chAPE1) gene was cloned a few years ago [37]. We
cloned the cDNA of chAPE1, expressed it in insect cell

line sf9 and examined the activity of the enzyme in vi-
tro. We investigated the levels of the two main cata-
lytic activities, 3¢–5¢ exonuclease activity and the
Fig. 4. Phosphorylation effects on the APE activity of chAPE1. The
rate constant K
obs
was calculated using the equation
ln([S
t
] ⁄ [S
o
]) = )K
obs
*t for first or pseudo-first order reactions. 24 h:
chAPE1 expressed at the 24 h time point. CK I, CK II, LP and CIP:
chAPE1 activity treated with CK I, II, lambda phosphatase and alka-
line phosphatase, respectively. CK I + LP, CK II + LP: chAPE1 trea-
ted with CK I or CK II first and then dephosphorylated with lambda
phosphatase before the activity assay. CK II decreased the chAPE1
activity level by 6.2-fold and lambda phosphatase increased the
activity by 2.1-fold, and also restored the activity level of CK II-inhib-
ited chAPE1.
AB
Fig. 5. Endonuclease activity of chAPE1.
(A) Michaelis–Menten analysis of the activity
of chAPE1 phosphorylated by CK II
with k
cat
⁄ K
m

= 7.2 · 10
)3
nM
)1
Æs
)1
.
(B) Michaelis–Menten analysis of the activity
of chAPE1 dephosphorylated by lambda
phosphatase previously phosphorylated by
CK II, k
cat
⁄ K
m
= 0.12 nM
)1
Æs
)1
.
Fig. 6. Exonuclease activity of chAPE1 and exonuclease III (autora-
diograph of the gel image). 100 n
M of double-stranded DNA oligo
substrate (5¢-GTCACCGTCATACGACTC-3¢, complementary strand
was not shown, and both strands were labeled with P
33
isotope),
100 n
M chAPE1 and 10 nM Escherichia coli exonuclease III as a
control were used in this particular assay.
M. Borjigin et al. Phosphorylation controls chAPE1 activity

FEBS Journal 277 (2010) 4732–4740 ª 2010 The Authors Journal compilation ª 2010 FEBS 4735
endonuclease activity. We did not detect exonuclease
activity under our experimental conditions. Endonucle-
ase activity varied from batch to batch, but remained
within the broad range obtained for other mammalian
APE1 enzymes and especially human APE1 [13–18].
However, activity varied with the time of the culture.
In order to resolve this variation, we investigated our
hypothesis that the activity of the enzyme can be
controlled by phosphorylation. Indeed, we detected
such a control and were able to narrow the range of
possible phosphatases and kinases that can potentially
control it.
The enzyme kinetic assays performed on chAPE1
phosphorylated by CK II and dephosphorylated by
lambda phosphatase using a Michaelis–Menten model
revealed the kinetic parameter values of chAPE1. The
phosphorylation efficiencies of both CK I and CK II
on chAPE1 were fully comparable (data not shown)
with that previously reported [38]. We observed that
CK I had no effect on chAPE1 endonuclease activity,
which is consistent with the findings of Yacoub et al.
[38]. However, the level of inhibition of chAPE1 activ-
ity by CK II is not as complete as that of the human
APE1 activity reported in [38]. The difference might be
attributed to the species or expression host difference.
Alkaline phosphatase did not alter the activity level of
the phosphorylated chAPE1, whereas lambda phos-
phatase increased the catalytic activity of chAPE1.
These data imply that CK I and CK II target different

amino acids in chAPE1 and lambda phosphatase may
work on a common set of amino acid residues with
CK II.
In particular, we were interested in the similarities
and differences to the human APE1, for which signifi-
cantly divergent activities have been reported. The evi-
dence accumulated over many years shows that
phosphorylation regulates the human APE1 endonu-
clease and redox activities in vitro [38–40]. Several
groups have investigated the effects of phosphoryla-
tion on the endonuclease and redox activities of
human APE1. Although the results of their reports
were somewhat conflicting, the consensus conclusion
was that phosphorylation regulates the two important
functions of human APE1 [38–40], which might switch
on and off different functions at different physiologi-
cal conditions. Although the phosphorylation of
human APE1 by CK II was reported to have no
effect [39] to complete inhibition [38] on its APE
activity, redox activity was enhanced by CK II treat-
ment [39]. In addition to the in vitro evidence of the
regulation of human APE1 endonuclease activity by
kinases, a very recent report showed that human
APE1 is phosphorylated by another regulatory pro-
tein, cdk5, which reduces the endonuclease activity of
APE1. This in vivo experiment carried out on mice
demonstrated that phosphorylation of APE1 at Thr
232 reduces its APE activity, resulting in an accumula-
tion of DNA damage and contributing to neuronal
death [12]. The increased phosphorylation of APE1

was also observed in post-mortem brain tissue from
patients with Parkinson’s disease and Alzheimer’s dis-
ease, suggesting a potential link between APE1 phos-
phorylation and the pathogenesis of neurodegenerative
diseases [12].
In our study of the phosphorylation of chAPE1, we
determined quantitatively the extent of phosphoryla-
tion by using a novel and accurate analytical tool. We
were fortunate to have access to state of the art equip-
ment, an ICP spectrometer, an instrument with pico to
nanomolar sensitivity in measuring trace elements from
aqueous solutions. We were successful in measuring
the absolute amount of phosphorus and sulfur from
micromolar protein samples with significant accuracy.
Similar measurements were carried out with this
instrument to determine the same elements in vegetable
oils and beef [41] and other trace elements bound to
proteins [42]. This technique offers significant advanta-
ges over traditionally used methods.
There are several more traditional methods of direct
measurement of phosphorylation. The most popular
involves the incubation of whole cells with radiola-
beled
32
P-orthophosphate, the generation of cellular
extracts, separation of proteins by SDS ⁄ PAGE and
exposure to film for phosphoimaging [43]. A clear
drawback of this method is labor expense and the use
of radioactive isotopes and the difficulty of eliminat-
ing the background presence of a natural phosphate

source in the culture medium. Another specific tech-
nique is the use of phosphate-specific antibodies. This
technique can be used for an immunoassay to deter-
mine the phosphorylation amount [44]. The main
caveat in successfully utilizing a phosphor-specific
antibody technique is the specificity and affinity of the
antibody for the phosphoprotein of interest. The most
accurate and powerful technique for determining and
sequencing the phosphoproteins is MS [45]. However,
there are also several difficulties with the analysis of
phosphoproteins by this technique. First, signals from
phosphopeptides are generally weaker, as they are
negatively charged and poorly ionized by electrospray
MS (it is performed in the positive mode). Second, it
can be difficult to observe the signals from low-abun-
dance phosphoproteins of interest in the high back-
ground of abundant nonphosphorylated proteins [45].
One of the more innovative uses of this technique was
developed by McKenzie & Strauss [46]. However, this
Phosphorylation controls chAPE1 activity M. Borjigin et al.
4736 FEBS Journal 277 (2010) 4732–4740 ª 2010 The Authors Journal compilation ª 2010 FEBS
technique was used to measure the phophorylation
efficiency of kinases with a radioactive phosphate
compound.
In summary, the level of phosphorylation of
chAPE1 in sf9 cells varied at different time points of
expression, which correlated well with its endonuclease
activity. The observation was confirmed by a kinetic
assay of the phosphorylated and dephosphorylated
chAPE1. The in vitro catalytic activity tests also dem-

onstrated that different regulatory proteins (kinases
and phosphatases) have different effects on chAPE1.
These results suggest that the different functions of the
multifunctional chAPE1 are switched on and off by
regulatory proteins at different stages of the cell life,
which might also provide a plausible explanation for
the reported discrepancies in endonuclease activity
level of the human APE1 in the literature. Our final
finding suggests that chAPE1 may not have exonucle-
ase activity regardless of its phosphorylation state.
This implies that further studies into exonuclease activ-
ity of human APE1 in the phosphorylated and ⁄ or
dephosphorylated state are warranted. This important
regulatory effect of phosphorylation has not been
explored exhaustively in other mammalian APE1.
More studies in this area with human enzyme are also
warranted.
Experimental procedures
Overexpression of chAPE1 in the sf9 cell line
using the baculovirus system
The cDNA developed in our laboratory was subcloned into
a baculovirus transfer vector (pBlueBacHis2B; Invitrogen,
Carlsbad, CA, USA) using PCR amplification with a
pair of designed primers. The sequences of the primers
were 5¢-GAAGATCTAAGCGTGGGAAGAGAGCG-3¢
and 5¢-GGGGTACCAGGTGTAAGTTACTTCAGCAG-3¢
(MWG Biotech, Ebersberg, Germany). The insect cell line
sf9 was cotransfected with the pBlueBacHis2B construct
and Bac-N-Blue AcMNPV viral DNA (Invitrogen), fol-
lowed by an agarose overlay plaque assay to select the

recombinant virus. High titer virus stocks (up to 1.3 · 10
8
plaque forming unitsÆmL
)1
) were generated in a suspension
sf9 cell culture. Large-scale protein expression was carried
out in 500 mL suspension culture with viral stock at a mul-
tiplicity of infection of eight.
The cells were harvested and lysed by sonication followed
by centrifugation. The supernatant was applied to Ni-NTA
affinity and S75 Sepherose size exclusion columns to purify
the protein under native conditions. The N-terminal
( 4 kDa) HisTag linker was cleaved with enterokinase
(EKmax; Invitrogen) and the native chAPE1 was isolated
from the Tag and enterokinase using a nickel affinity
column and an EKaway resin column. Protein purity was
tested using SDS ⁄ PAGE followed by Coommassie blue
staining; the concentration was determined using the Brad-
ford protein assay (Bio-Rad, Hercules, CA, USA).
Initial endonuclease activity screen
Fifty milliliters of sf9 cell culture were removed from
500 mL suspension culture at 24, 48 and 72 h postinfection
with the recombinant baculovirus. chAPE1 was purified
and quantified from the 50 mL cell culture aliquots, using
the same procedure as described above. The enzyme at
three time points of expression from three different batches
of culture was prepared. An abasic DNA substrate was made
by annealing a 5¢ P
33
-labeled, tetrahydrofuran-containing

oligonucleotide (5¢-GTCACCGTC
FTACGACTC-3¢) with
its complementary oligonucleotide (MWG Biotech) in
50 mm HEPES buffer, pH 7.5, 50 mm KCl, 0.1 m m EDTA
by heating in a 95 °C water bath and cooling down at
room temperature within 2 h.
The endonuclease reaction was carried out in a total vol-
ume of 30 lL containing 100 nm DNA substrate and 5 nm
chAPE1 in 50 mm HEPES ⁄ KOH (pH 7.5), 50 mm KCl,
0.1 mm EDTA and 5 mm MgCl
2
at room temperature [26].
Aliquots of 3 lL of reaction mix were transferred into 3 lL
stop solution containing 85 mm EDTA at designated time
points to quench the reaction. Subsequently, the reaction
products were resolved in a DNA sequencing gel (15%
polyacrylamide gel containing 8 m urea). The gels were
dried, exposed to K screen (BioRad) and the image and the
band intensities measured and quantified with a BioRad
PhosphoImager FX and quantity one software. All
enzyme kinetic experiments were repeated in triplicate
and K
obs
was calculated using the formula ln([S
t
] ⁄ [S
o
]) =
)K
obs

*t, which applies to first or pseudo-first order reac-
tions. Here, [S
o
]=[S
t
]+[P
t
]; [S
t
] is the uncleaved (or
intact) substrate concentration, [P
t
] is the product concen-
tration at time point t,[S
o
] is the initial (or total) substrate
concentration. The quantitation proceeded through measur-
ing the intensity of both bands and normalizing them by
adding both bands and taking the fractional intensity
belonging to the particular (substrate, product) band. Such
measured intensity of bands was used to construct K
obs
plots and obtaining full Michaelis–Menten substrate satura-
tion curves.
Steady-state kinetic studies on the endonuclease
activity of chAPE1 and Michaelis–Menten
analysis
In order to determine the kinetic parameters of the endonu-
clease activity of chAPE1, 5 nm chAPE1 was mixed with
various concentrations (10–400 nm) of abasic DNA

M. Borjigin et al. Phosphorylation controls chAPE1 activity
FEBS Journal 277 (2010) 4732–4740 ª 2010 The Authors Journal compilation ª 2010 FEBS 4737
substrate in 30 lL of reaction under the standard condi-
tion, and the subsequent procedures of the assay were the
same as described above. The Michaelis–Menten analysis
was carried out with the SigmaPlot enzyme kinetic module
to calculate the parameter values.
ICP analysis to measure phosphate amount
Because the sensitivity of ICP for sulfur and phosphorus
is in the subnanomolar scale, it is quite appropriate to
measure accurately the amounts of these elements in a
protein sample of micromolar concentration. An Optima
4300 DV ICP spectrometer (Perkin Elmer, Boston, MA,
USA) was used with the following parameters: nebulizer
backpressure 258.0 kPa, nebulizer flow 0.80 LÆmin
)1
, wave-
lengths for P 213.617 nm, S 180.669 nm. The nine protein
preparations from three time points of three different
batches of chAPE1 were dialyzed in 50 mm Tris ⁄ HCl, pH
8.0, and their concentration adjusted to 250 lgÆ mL
)1
in
3 mL volume. The standard curve was constructed with
solutions of known concentration of phosphorus and
sulfur and other elements for references, in parts per
billion concentration. The absolute concentration of sulfur
and phosphorus was determined based on the conversion
of the spectrum intensity to a concentration value. The
phosphorus in the protein samples was accurately

calculated using the ratio of the molarity of the protein
and the measured molarity of phosphorus, and sulfur was
used as the reference control for the calculation. Each
sample was measured in triplicate and the error was
calculated using Microsoft Excel.
Phosphorylation of chAPE1 with CK I or CK II
Both kinases and buffers were obtained from New England
Biolabs (Beverly, MA, USA). Five pmol of chAPE1 was
phosphorylated in a 50 lL reaction volume using 5 units of
CK I or CK II. The CK II reaction condition was 0.1 mm
ATP, 0.6 lCiÆlL
)1
[c
)33
P]ATP, 20 mm Tris ⁄ HCl (pH 7.5),
50 mm KCl and 10 mm MgCl
2
. The CK I reaction condition
was 0.1 m m ATP, 0.6 lCiÆlL
)1
[c
)33
P]ATP, 50 mm
Tris ⁄ HCl (pH 7.5), 5 mm dithiothreitol and 10 mm MgCl
2
.
The reaction mix was incubated at 30 °C for 45 min, and the
efficiency of phosphorylation was assessed in SDS ⁄ PAGE
followed by imaging and quantification by means of BioRad
PhosphoImager FX and quantity one software.

Dephosphorylation of chAPE1 with alkaline
phosphatase or lambda phosphatase
chAPE1 (2 pmol) was dephosphorylated using alkaline
phosphatase (0.5 unit) or lambda phosphatase (5 units) in
20 lL reaction mix at 30 °C for 45 min. The alkaline phos-
phatase reaction was carried out in 100 mm NaCl, 50 mm
Tris ⁄ HCl (pH 7.9), 10 mm MgCl
2
and 1 mm dithiothreitol.
The conditions for lambda phosphatase were 50 mm
Tris ⁄ HCl (pH 7.5), 0.1 mm Na
2
EDTA, 5 mm dithiothreitol,
0.01% Brij 35 surfactant and 2 mm MnCl
2
.
Exonuclease assay
The standard exonuclease assay was conducted using the
same procedures as for the APE activity, except for the reg-
ular double-stranded DNA oligo substrate, of which the 5¢
of each strand was labeled with the P
33
isotope. The
concentration of the enzymes (100 nm chAPE1 and 10 nm
Escherichia coli exonuclease III) and 10–400 nm DNA
substrate were used. Here, Escherichia coli exonuclease III
(New England Biolabs) was used as a positive control.
Acknowledgements
We thank Dr Siddhartha Das for helpful discussion on
the subjects and thank Drs Jorge Gardea-Torresdey

and Jose Peralta for their help in performing experi-
ments using the ICP and providing necessary
reagents. This work was partially supported by NIH
grants GM08012, RR008124 and the NSF grant
HRD9701775 to Dr P. Arenaz.
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