chinese hamster apurinic/apyrimidinic endonuclease (chape1) expressed in sf9 cells reveals that its...

Chinese hamster apurinic ⁄apyrimidinic endonuclease(chAPE1) expressed in sf9 cells reveals that itsendonuclease activity is regulated by phosphorylationMandula Borjigin1, Bobbie Martinez2, Sarla Purohit2, Gaudalupe de la Rosa2, Pablo Arenaz2 andBoguslaw Stec3

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: [email protected]

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: [email protected]

(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¢ toan 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¢-repairphosphodiesterase 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 Km ranges from 3.4 to

200 nm, kcat from 1.38 to 10 s)1 and kcat ⁄Km from 0.05

to 0.5 nm)1Æs)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¢ exonucleaseactivities 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 Kobs 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 (Kobs) 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 R2 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.


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 (kcat ⁄Km) by 16.7-fold relative to the

activity of chAPE1 phosphorylated by CK II. The

kinetic parameters for chAPE1 phosphorylated by CK

II were kcat = 0.58 ± 0.02 s)1, Km = 81 ± 10.59 nm

and kcat ⁄Km = 7.2 · 10)3 nm)1Æs)1. The parameter

values for chAPE1 dephosphorylated by lambda

phosphatase were kcat = 5.67 ± 0.21 s)1, Km = 48 ±

6.98 nm and kcat ⁄Km = 0.12 nm)1Æs)1 (Fig. 5). These

results, along with the Kobs 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 Kobs was calculated using the equation

ln([St] ⁄ [So]) = )Kobs*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.

A B

Fig. 5. Endonuclease activity of chAPE1.

(A) Michaelis–Menten analysis of the activity

of chAPE1 phosphorylated by CK II

with kcat ⁄ Km = 7.2 · 10)3 nM)1Æs)1.

(B) Michaelis–Menten analysis of the activity

of chAPE1 dephosphorylated by lambda

phosphatase previously phosphorylated by

CK II, kcat ⁄ Km = 0.12 nM)1Æs)1.

Fig. 6. Exonuclease activity of chAPE1 and exonuclease III (autora-

diograph of the gel image). 100 nM of double-stranded DNA oligo

substrate (5¢-GTCACCGTCATACGACTC-3¢, complementary strand

was not shown, and both strands were labeled with P33 isotope),

100 nM chAPE1 and 10 nM Escherichia coli exonuclease III as a

control were used in this particular assay.



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 32P-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



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 ⁄ordephosphorylated 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 · 108

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¢ P33-labeled, tetrahydrofuran-containing

oligonucleotide (5¢-GTCACCGTCFTACGACTC-3¢) with

its complementary oligonucleotide (MWG Biotech) in

50 mm HEPES buffer, pH 7.5, 50 mm KCl, 0.1 mm 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 MgCl2 at room temperature [26].

Aliquots of 3 lL of reaction mix were transferred into 3 lLstop 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 Kobs was calculated using the formula ln([St] ⁄ [So]) =

)Kobs*t, which applies to first or pseudo-first order reac-

tions. Here, [So] = [St] + [Pt]; [St] is the uncleaved (or

intact) substrate concentration, [Pt] is the product concen-

tration at time point t, [So] 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 Kobs

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



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 lgÆmL)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)33P]ATP, 20 mm Tris ⁄HCl (pH 7.5),

50 mm KCl and 10 mm MgCl2. The CK I reaction condition

was 0.1 mm ATP, 0.6 lCiÆlL)1 [c)33P]ATP, 50 mm

Tris ⁄HCl (pH 7.5), 5 mm dithiothreitol and 10 mm MgCl2.

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 MgCl2 and 1 mm dithiothreitol.

The conditions for lambda phosphatase were 50 mm

Tris ⁄HCl (pH 7.5), 0.1 mm Na2EDTA, 5 mm dithiothreitol,

0.01% Brij 35 surfactant and 2 mm MnCl2.

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 P33 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.

References

1 Evans AR, Limp-Foster M & Kelley MR (2000) Going

APE over ref-1. Mutat Res 461, 83–108.

2 Manoharan M, Mazamder A, Ransom SC, Gerlt JA &

Bolton PH (1988) Mechanism of UV endonuclease V

cleavage of abasic sites determined by 13C labeling.

J Am Chem Soc 110, 2690–2691.

3 Taylor AF & Weiss B (1982) Role of exonuclease III in

the base excision repair of uracil-containing DNA.

J Bacteriol 151, 351–357.

4 Schaaper RM & Leob LA (1981) Depurination causes

mutations in SOS-induced cells. Proc Natl Acad Sci

USA 78, 1773–1777.

5 Shearman CW & Leob LA (1977) Depurination

decreases fidelity of DNA synthesis in vitro. Nature 270,

537–538.

6 Wilson DM III & Thompson LH (1997) Life

without DNA repair. Proc Natl Acad Sci USA 94,

12754–12757.

7 Lindahl T, Karrans P & Wood R (1997) DNA excision

repair pathways. Curr Opin Genet Dev 7, 158–169.

8 Richardson C & Kornberg A (1964) A deoxyribonucleic

acid phosphatase-exonuclease from Escherichia coli. I.

Purification of the enzyme and characterization of the

phosphatase activity. J Biol Chem 239, 242–250.

9 Richardson C, Lehman I & Kornberg A (1964)

A deoxyribonucleic acid phosphatase-exonuclease from



Escherichia coli. II. Characterization of the exonuclease

activity. J Biol Chem 239, 251–258.

10 Demple B, Johnson A & Fung D (1986) Exonuclease

III and endonuclease IV remove 3¢ blocks from DNA

synthesis primers in H2O2-damaged Escherichia coli.

Proc Natl Acad Sci USA 83, 7731–7735.

11 Bernelot-Moens C & Demple B (1989) Multiple DNA

repair activities for 3¢-deoxyribose fragments in Escheri-

chia coli. Nucleic Acids Res 17, 587–600.

12 Huang E, Qu D, Zhang Y, Venderova K, Haque ME,

Rousseaux MW, Slack RS, Woulfe JM & Park DS

(2010) The role of Cdk5-mediated apurinic ⁄ apyrimidinic

endonuclease 1 phosphorylation in neuronal death. Nat

Cell Biol 12, 563–571.

13 Wilson DM III, Takeshita M, Grollman AP & Demple

B (1995) Incision activity of human apurinic endonucle-

ase (Ape) at abasic site analogs in DNA. J Biol Chem

270, 16002–16007.

14 Mol CD, Izumi T, Mitra S & Tainer JA (2000) DNA-

bound structures and mutants reveal abasic DNA bind-

ing by APE1 DNA repair and coordination. Nature

403, 451–456.

15 Mckenzie JA & Strauss PR (2001) Oligonucleotides

with bistranded abasic sites interfere with substrate

binding and catalysis by human apurinic ⁄ apyrimidinic

endonuclease. Biochemistry 40, 13254–13261.

16 Strauss PR, Beard WA, Patterson TA & Wilson SH

(1997) Substrate binding by human apurinic ⁄ apyrimidi-

nic endonuclease indicates a Briggs-Haldane mecha-

nism. J Biol Chem 272, 1302–1307.

17 Lucas JA, Masuda Y, Bennett RAO, Strauss NS &

Strauss PR (1999) Single-turnover analysis of mutant

human apurinic ⁄ apyrimidinic endonuclease. Biochemis-

try 38, 4958–4964.

18 Izumi T, Schein CH, Oezguen N, Feng Y & Braun W

(2004) Effects of backbone contacts 3¢ to the abasic site

on the cleavage and the product binding by human apu-

rinic ⁄ apyrimidinic endonuclease (APE1). Biochemistry

43, 684–689.

19 Yoshita A & Ueda T (2003) Human AP endonuclease

possesses a significant activity as major 3¢–5¢ exonucle-ase in human leukemia cells. Biochem Biophys Res Com-

mun 310, 522–528.

20 Chou K & Cheng Y (2002) An exonucleolytic activity

of human apurinic ⁄ apyrimidinic endonuclease on

3¢ mispaired DNA. Nature 415, 655–659.

21 Barzilay G, Walker LJ, Robson CN & Hickson ID

(1995) Site-directed mutagenesis of the human DNA

repair enzyme HAP1: identification of residues impor-

tant for AP endonuclease and RNase H activity.

Nucleic Acids Res 23, 1544–1550.

22 Suh D, Wilson DM III & Povirk LF (1997) 3¢-phospho-diesterase activity of human apurinic ⁄ apyrimidinic

endonuclease at DNA double-strand break ends.

Nucleic Acids Res 25, 2495–2500.

23 Demple B, Herman T & Chen DS (1991) Cloning and

expression of APE, the cDNA encoding the major

human apurinic endonuclease: definition of a family of

DNA repair enzymes. Proc Natl Acad Sci USA 88,

11450–11454.

24 Robson CN & Hickson ID (1991) Isolation of cDNA

clones encoding a human apurinic ⁄ apyrimidinic endo-

nuclease that corrects DNA repair and mutagenesis

defects in E. coli xth (Exonuclease III) mutants. Nucleic

Acids Res 19, 5519–5523.

25 Lebedeva NA, Khodyreva SN, Favre A & Lavrik OI

(2003) AP endonuclease 1 has no biologically significant

3¢-5 exonuclease activity. Biochem Biophys Res Commun

300, 182–187.

26 Seki S, Ikeda S, Watanabe S, Hatsushika M, Tsutsui K,

Akiyama K & Zhang B (1991) A mouse DNA repair

enzyme (APEX nuclease) having exonuclease and apuri-

nic ⁄ apyrimidinic endonuclease activities: purification

and characterization. Biochim Biophys Acta 1079, 57–

64.

27 Seki S, Akiyama K, Watanabe S, Hatsushika M, Ikeda

S & Tsutsui K (1991) cDNA and deduced amino acid

sequence of a mouse DNA repair enzyme (APEX

nuclease) with significant homology to Escherichia coli

exonuclease III. J Biol Chem 266, 20797–20802.

28 Robson CN, Milne AM, Pappin DJC & Hickson ID

(1991) Isolation of cDNA clones encoding an enzyme

from bovine cells that repair oxidative DNA damage

in vitro: homology with bacterial repair enzyme. Nucleic

Acids Res 19, 1087–1092.

29 Huq I, Wilson TM, Kelley MR & Deutsch WA (1995)

Expression in Escherichia coli of a rat cDNA encoding

an apurinic ⁄ apyrimidinic endonuclease. Mutat Res 337,

191–199.

30 Berquist BR, Singh DK, Fan J, Kim D, Gillenwater E,

Kulkarni A, Bohr VA, Ackerman EJ, Tomkinson AE

& Wilson DM III (2010) Functional capacity of

XRCC1 protein variants identified in DNA repair-defi-

cient Chinese hamster ovary cell lines and the human

population. Nucleic Acids Res 38, 5023–5035.

31 Pan Y, Yuan D, Zhang J, Xu P, Chen H & Shao C

(2009) Cadmium-induced adaptive response in cells of

Chinese hamster ovary cell lines with varying DNA

repair capacity. Radiat Res 171, 446–453.

32 Biverstal A, Johansson F, Jenssen D & Erixon K (2008)

Cyclobutane pyrimidine dimers do not fully explain the

mutagenicity induced by UVA in Chinese hamster cells.

Mutat Res 648, 32–39.

33 Taccioli GE, Cheng H-L, Varghese AJ, Whitmore G &

Alt FW (1994) A DNA repair defect in Chinese hamster

ovary cells affects V(D)J recombination similarly to the

murine mid mutation. J Biol Chem 269, 7439–7442.

34 Boiteux S & le Page F (2001) Repair of 8-oxoguanine

and Ogg1-incised apurinic sites in a CHO cell line. Prog

Nucleic Acid Res Mol Biol 68, 95–105.



35 Gille JJ, van Berkel CG & Joenje H (1993) Mechanism

of hyperoxia-induced chromosomal breakage in Chinese

hamster cells. Environ Mol Mutagen 22, 264–270.

36 Lehmann AR (1985) Use of recombinant DNA tech-

niques in cloning DNA repair genes and in the study of

mutagenesis in mammalian cells. Mutat Res 150, 61–67.

37 Purohit S & Arenaz P (1999) Molecular cloning,

sequence and structure analysis of hamster apuri-

nic ⁄ apyrimidinic endonuclease (chApel) gene. Mutat

Res 435, 215–224.

38 Yacoub A, Kelley M & Deutsch WA (1997) The DNA

repair activity of human redox ⁄ repair APE ⁄Ref-1 is

inactivated by phosphorylation. Cancer Res 57, 5457–

5459.

39 Fritz GB & Kina B (1999) Phosphorylation of the

DNA repair Ape1 ⁄ ref1 by CK II affects redox regula-

tion of AP-1. Oncogene 18, 1033–1040.

40 Hsieh MM, Hegde V, Kelley MR & Deutsch WA

(2001) Activation of APE ⁄Ref-1 redox activity is medi-

ated by reactive oxygen species and PKC phosphoryla-

tion. Nucleic Acids Res 29, 3116–3122.

41 Ostrovsky M, Loukianova T, Hilligoss D & Wee P.

Sulphur and Phosphorus Analysis in Vegetable Oil and

Beef Tallow for Biodiesel Production Using the Optima

Inductively Coupled Plasma-Optical Emission Spectrome-

ter, http://las.perkinelmer.com/FocusAreas/Mkt+Sus-

tainable+Energy/Biofuels_Application_Notes.htm.

42 Ma R, McLeod CW, Tomlinson K & Poole RK (2004)

Speciation of protein-bound trace elements by gel elec-

trophoresis and atomic spectrometry. Electrophoresis

25, 2469–2477.

43 de Graauw M, Hensbergen P & van de Water B (2006)

Phospho-proteomic analysis of cellular signaling. Elec-

trophoresis 27, 2676–2686.

44 Czernik AJ, Girault JA, Nairn AC, Chen J, Snyder G,

Kebabian J & Greengard P (1991) Production of phos-

phorylation state-specific antibodies. Methods Enzymol

201, 264–283.

45 Mann M, Ong SE, Gronborg M, Steen H, Jensen ON

& Pandey A (2002) Analysis of protein phosphorylation

using mass spectrometry: deciphering the phosphoprote-

ome. Trends Biotechnol 20, 261–268.

46 Mckenzie JA & Strauss PR (2003) A quantitative

method for measuring protein phosphorylation. Anal

Biochem 313, 9–16.



chinese hamster apurinic/apyrimidinic endonuclease (chape1) expressed in sf9 cells reveals that its...

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