a mouse dna repair enzyme (apex nuclease) having exonuclease and apurinic/apyrimidinic endonuclease...

Biochit, lica et Biophysica Acta, 1079 (19ql) 57-64 © 1991 Elsevier Science Publishers B.V. 0167-4838/ql/$03.50 ADONIS 0167483891002536

BBAPRO 33980

A mouse DNA repair enzyme (APEX nuclease) having exonuclease and apurinic/apyrimidinic endonuclease activities:

purification and characterization

Shuji Seki, Shogo Ikeda *, Sekiko Watanabe, Masao Hatsushika, Ken Tsutsui, Kosuke Akiyama and Bo Zhang

Department of Molecular Biology. Institute o[ Cellular and Molecular Biolo~', Okayama Uniuersiry Medical School. Okayama ¢Japan)

(Received 18 March 1991)

57

Key words: DNA repair enzyme; Exonuclease: Apurinic/apyrimidinic endoauclease; DNA phosphatase; Amino acid sequc0ce; Priming enzyme

A mouse repair enzyme having priming activity on bleomycin-damaged DNA for DNA polymerase was purified to apparent homogeneity and characterized. The enzyme extracted from permeabilized mouse ascites sarcoma (SR-C3H/He) cells with 0.2 M potassium phosphate buffer (pH 7.5) was purified by successive chramatograpbies on phosphocellulose, DEAE-cellulose, phosphocellulose (a second time), ~phadex G.100, single.stranded DNA cellulose and hydroxyapatite. The purified enzyme has an M, of 34000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Enzymatical studies indicated that it is a multifunctional enzyme having exonuclease, apurinic/apyrimidinic endonuclease and phosphatase activities, similar to Escher/ch/a co/i exonuclease !I!. This enzyme is tentatively designated as APEX nuclease for apurinic/apyrimidinic endonuclease and exonuclease activities. The amino acid composition, amino-terminal amino acid sequence and an internal amino acid sequence of APEX nuclease are determined.

Introduction

The DNA damage that occurs daily as a conse- quence of spontaneous hydrolysis under physiological conditions or by chemical, physical or biological stresses is substantial and of various types [I]. Among the types of damage, apurinic/apyrimidinic (AP} sites are gener- ated at several thousand residues per genome per day in a mammalian cell [2]. Single-strand DNA breaks with 3' termini blocked I%' nucleotide fragments are also known to be frequently induced by free radical pathways initiated by ionizing radiation, bleomycin and other sources of oxygen radicals [1,3-6]. In our studies on DNA repair synthesis after bleomycin-induced dam-

* Present address: Department of Bu~logical Chcmi,try, Facully ¢~[ Science. Okayama Univer~a~ of Science. Okayama 7(10. ~'apan.

Correspondence: S. Seki. Department of Molecular BioloK,. Insti- tute of Cellular and Molecular Biolog'y, Oka~ama Universily Medical School. 2-5-1. Shikata-cho. Okayama 711~). Japan

age in mammalian cells, we found and partially purified a priming enzyme (an exonuclease) that can initi- ate repair of bleomycin-induced and X-ray-induced single-strand breaks [7-11]. This enzyme also showed priming activiD' for DNA polymerase on acid-depurinated DNA and micrococcal nuclease-treated DNA, .suggesting that it is a multifunctional enD'rae having exonuclease. AP endonuclease and phosphatase act vities [9.12]. Thus this enzsme exhibits properties sir dlar to those of E. coli ,:xonuclea~ !I1 and yeast DNA 3'-repair diesterase [IA3,1a]. Mammalian AP endonucleases possibly involved in the repair of AP sites have been purified from various species including mice [15-171. rats [181, cattle [t9] and humans [20-221. As far as we know, however, none of the previously reported mammalian AP endonucleases possessed exonuclease actMty.

In the present paper we describe the purification, characterization and partial amino acid sequences of the mouse repair enzyme (designated as APEX nuele- ase) having exonuclease and AP endonuclease activities.

58

Materials and Methods

Materials The reagents used in these experiments were ob-

tained from the following sources: [~H]dTTP from Amersham Japan, Tokyo, Japan: [a~'P]phos~hate and [a-3-'P]dCTP from ICN Biochemicals, CA, U.S.A.; ribonucleotides {NTPs) and deoxyribonuclcotides (dNTPs) from Seikagakv. Kngyou, Tokyo, Jap,m: E. colt exonuclease Ill, DNA polymerase I and the large fragment (Klenow polymcrase) of DNA polymerase I from Takara Shuzo. Kyoto, Japan; calf thymus DNA and DNA alternate copolymer poly(dA-dT)-poly(dA- dT) from Pharmacia, Uppsala, Sweden. The other reagents used were obtained from the previously described sources [Q]. Mouse ascites sarcoma [SR- C3H/He) cells were obtained and maintained as described previously [23]. pUC18 DNA was prepared as described previously [2,1]. Bleomycin-Fc(ll)-trcated ¢calf thymus and pUCI8) DNAs and X-irradiated DNA were preparcd as described previously [9-11].

Preparation of acid-depurinated DNA and assay for AP endonuclease actie i.,y

The superhelical (form 1) pUCIS DNA and calf thymus DNA were depurinated by i~lcubating them in 3 vol. of 50 mM sodium citrate (pH 3.5) at 60°C for 15 rain essentially as described by Niwa and Moses [9.25]. After the incubation, the mixture was chilled to 0°C and dialysed against 50 mM Tris-HCI (pH 7.5) for 3 h and t,en against distilled water overnight. The dialysed DNA solution was used to measure AP endonucleasc activity. The acid treatment produced approximately six alkali-sensitive sites per pUCI8 DNA molecule, as determined by alkaline gel eleetrophoresis [26]. The assay mixture (15 tal final vol.) co:retained 0.25 ~,g (0.14 pmol) acid-depurinated pUCI8 DNA and 0-7.5 ~tl of an appropriate dilution of the enzyme in Triton-buffer B (0.0175% Triton X-ID0. 0.25 M sucrose, 10 mM Tris-HCl, 4 mM MgCI:, [ mM EDTA and 6 mM 2-mereaptoethano[, pH 8JI adjusted at 25°C1. After incubating the assay raixture at 37°C for 20 rain, the reaction was stopped by chilling it to 0°C and then adding 3 tal of 6-fold-concentrated gel loading buffer (0.25% bromphenol blue, 0.25% xylenc cyanol and 30% glycerol in H20). The mixture was loaded into a slot of a submerged 0.8% agarose gel. Electrophoretic analyses of conformation of pUC18 DNA were conducted as described previously [10,11,24].

Assay of the enzyme that enhances the template-primer actit'i~" of bleomycin-damaged, X-irradiated or acid.depurinated DNA for DNA polymemse 13 or Klenow polymerase

In the present paper the term "template-primer activity' of DNA is used to indicate the ability to ser~c as

a template-primer for DNA polymerase /~ or Klenow polymerase. The term 'priming activity (or enzyme)" on bteomycin-damaged, X-irradiated or acid-depurinated DNA is used to denote the activity (or enzyme) that enhances the template-primer activity of bleomycin- damaged, X-irradiated or acid-dcpurinated DNA by removing the 3' tags or by introducing single-strand breaks. The priming activity was measured by the two- step- {priming and repair DNA synthesis step-} method [U, ll]. For measuring the priming activity on bleomycin-damaged DNA, for example, 6 tag calf thymus DNA was incubated at 37°C for 30 min with 20 #M Fe(ll), 0.2 ~.g bleomycin and an appropriate an~ount of a priming enzyme fraction in a 39-/~I reaction mixture made up with Tritor,-buffer B. After the incubation, the reaction mixture was incubated at 60°C fo~ I0 min to inactivate enzymes in the priming enzyme fraction and then chilled to 0°C. A 21-~1 aliquot of the substrate mixture (labeled substrate: [~H]dTTP) for DNA synthesis supplemented with 0.04 unit of Klenow polymerase or DNA polymerase /3 was added to the reaction mixture, DNA synthesis carried out at 37°C for 30 rain. The ractioactivity incorporated into acid-in- soluble materials was mcasured by a disc method [91. One unit of pri,ning enzyme in the present assay is defined as the amount of the enzyme causing 1 nmol of [3H]dTMP incorporation per 30 rain into bleomyein- damaged DNA under the standard assay conditions.

Assay of DNA double-stranded exonuclease and phosphatase acticitws

Radioactive DNA for exonuelease assays was prepared by growing Escherichia colt HBI01 in Luria- Bertani medium (125 ml) supplemented with [3H] thvmidine (i m C i / l ~ ml, 50 Ci/mmol). When the cclls reached the late log phase, they were harvested and their DNA was purified by standard phenol extrac- tion techniques. The radioactivit~ of the labeled DNA was 744 cpm/nmol of nucleotide. Bleomycin-, micrococcal nuclcase- or DNase l-treatment of the labeled DNA was conducted as described previously [9,27]. Exonuclease activity of the priming enzyme was measured using the bleomycin-, micrococcal nuclease- or DNasc l-treated, ~H-labeled DNA essentially as described [9]. To demonstrate 3'-5' exonuclease activity, the alternate copolymer poly(dA-dT) - poly(dA-dT) was 3'-end-labeled by incubating it with [3H]d'i~P and Klenow polymerase. The purified, 3'-end-labeled copolymers were incubated at 37°C for varying times with the priming enzyme in Triton-buffer B. Nu- cleotides released by exonucleolytic digestion were analyzed by polyethylene imine-cclluiose thin layer chromatography [28]. To measure DNA 3' phosphatase activity, 3'[3ZP]phosphoryl-terminated DNA (specific activity: 8.9.104 cpm/nmol of phosphate) was prepared by partially digesting ~:P-labeled E. colt DNA

5O

with micrococcal nucleasc [29,30]. The priming enzyme was incubated at 37°C with the 3'-[~:P]phosphor3l - terminated DNA and released [':P]inorganic phosphate (acid-soluble. Norit-non-adsorbable ":P) ~as measured as dcscribcd pre~4ously [31)].

Detennhmtion of partial amim~ acid sequences o[ the enzyme

When the purified priming enzyme 1,APEX nuclease) was treated at 37°C for 12.5 h with 70(~ formic acid. which hydrolyzes peptidc bonds between aspartate and proline, the enzyme was cleaved into two fragments with molecular masses of 30 and 4 kDa. ]'he amino-terminal amino acid sequence of the en~'me and that of the large formic acid fragment purified by SDS-polyaerylamide gel elcctrophoresis (SDS-PAGE) were determined by an Applied Biosystems Model 477A automated protein sequencer.

Other methods DNA polymerase/3 was purified [9,31] and assayed

[9] as described previously. Agmose gel electrophoresis and SDS-PAGE were performed as described previously [10-12,32]. Repair of pUCI8 DN,'~ with bleomycin-, peplomycin- or X-ray-induced single-strand breaks was measured by monitoring conformational changes of the plasmid DNA [10,11,26]. Protein con- centrations were determined by the BCA Protein As- say (Pierce, IIfinois. U.S.A), using bovine serum albumin as the standard. The amino acid composition of the enzyme was determined using an Applied Biosys- terns Derivatizer. Model 420A.

Results

Purification of a mouse repair enzj'me for bleomvcin- damaged DNA

Partial purification of a repair cnz,yme having exonucleolytic activity on bleomycin-damaged DNA and thereby providing priming sites for DNA polymerase was reported previously [9]. In the present stud}', the enzyme was further purified to apparent homogeneity. The purification was followed by measuring its DNA polymerase /3 prim!rig activity on bleomycin-damaged DNA. The enzyme was extracted from permeable mouse ascites sarcoma cells with 0.2 M potassium phosphate (KPi) buffer (pH 7.5) [9]. After adjustment of the KPi concentration to 0.1 M, the extract (fraction Nil was mixed with packed phosphtx;ellulose equilibrated with 0.1 M KPi. Enzymes bound to phosphocellulose were eluted with (;.3 M KPi. The eluant (fraction N 2) was passed through DEAE-celluk)se mainly to remove nuelcic acid as described previously 19,31]. Material (fraction N3) not retained by lhe DEAE-cellulose column was loaded onto tne ~cond phosphocellulose column equilibrated with 0.15 M KPi

J ~

, 3d.

10 [- J

0 13 30 40 50 60 70 FIq'ACT ION

8 MW 44 46 48 50 52 54 56 58 60

k D Q

2 9 ~ ~ ~ ~ - - ~ ~ -

2/.. ~. ~.- ~: . . . .

Fig I Second phosphocellukvse chromatogtzphs ot {he enzymc (priming IacIoL APEX n u c l e a r ) having DNA polvmerasc/~ primin~ actKily on bleomycin-dama~ed DNA. Panel A: the enzyme ,aas eluted ~tlh a linear gri~ienl eft potassium phosphate buffer (KPi. pit 75~ fmra IF 15 to 035 M. Priming acti',.t b v,a,, measured m the presence ( B t . M + . ~1 or absence (BLM - . .) cl bteom,F:m. :J- indl cared in Malerials and Methods The DNA ~x~L'~n3crase ~cttvtb ~1+ ot t:ach fraclion ",,,,as measured n,,ing aCli','~tcd DNA as a t,~mplale primer. Panel B: a pq~rti~m (33 ,all of eac,~ Waction (";(~ 44 6qi '.',a'., ei¢c~[,,lphorescd on a SI)S-Ix~t~a~.ryt-~mide gel and d~e gt:l ~as .,laJncd

~ l l h ( ' t~ma ' ~e brilhant blue R-25it M,Jtkcl pr<~tcin, b ~ i n c ~erur:, albumin (6S k D O chick ,~'.alhumm (43 kDa). t i the, nit ar~h,dra~c ~2 ~

kt)a) .and t~psmtrgen t24 kD:.O The 34-kDa prolcm b,md i, iadi cated b'. an az, lcri ,k

buffer (pH 7.5) containing 6 mM 2-mercaptoethanol. The protein was eluted with a linear gradient of KPi from 0.1. to I).35 M [9,31] and the enl}me cluted at (I.23 M kPi (Fig. lA). Analysis b} SDS-FA(;L show~ that the intensity of a 34-kDa protein band stained with Coomassie brilliant blue R-250 (CBB) correlates with the major peak of priming aetivi~ (Fig. IB). DNA polymerase/3 eluted slightly later than the major peak of priming activity. The fractions corresponJing to the major peak of priming activity (fraction N~' fractions 50-55 in Fig. lA) were collected and concentrated b.~ uhrafiltration using Centricut U-10 (molecular weight limit of filtration. 100011; Kurabo, Osaka. Japan). The concentrated sample was loaded onto a Sephadex G-

60

I(~) column and thcn cluted with TEMG (20 mM Tris-Cl (pH 8.01. I mM EDTA, 6 mM 2-mcrcapto- ethanol and 10% glycerol) supplemented with 11.2 M NaCI. The elution pattern of priming activity ano results of protcin analysis by SDS-PAGE are shown in Fig. 2A and B. The fractions (No. 82-1021 of the major peak N 5 w,:re pooled and then fractionatcd by single- strand DNA cellulose column chromatography as described previously [9.33]. The priming enzyme fractions (fraction N~,) eluted from the single-stranded DNA cellulose column were collected and dialyzed against PMG (0.1 M KPi, 6 mM 2-mercaptocthanol and 2115; glycerol). The dialyzed sample was Io'~ded onto a hy- dmxyapatite column equilibrated with PMG [33] ann the priming activity was eluted with a linear gradient of 0.1 M to 0.45 M potassium phosphate in PMG (Fig. 3A and B). The 34-kDa protein having the priming activity for repair DNA synthesis, fraction NT, was highly purified by this procedure.

';';" 15

~ 0

o

i i [ - ~ 1 t i

vo 6Ek 43k 24k 14k

60 70 80 90 100 ~tO ~20 133 140 FRACTION

MW 78

kDa

68 ~

43 ~

29~. .,..,. 2/.1~ -,,.,

02 86 90 9/. 98 102 106 110

Fig. 2. Sephadex G I l l 0 ct)luma chromatog!;!ph~ of the prtmmg

en~me. Panel A: fraction N 4 wa- c~mcentratcd and "~uhtecled to

Sephadcx G-1U0 c~aromatoglaph~ as d¢,,t,.bcd in the text. Priming

aclwity at each Iraction ~t)r DNA Ix~tymera~/3 was measured in t:~e

pr¢~nce (BLM -' . c;) or absence tBLM - . .1 of bleornycin. Panel V.:

a portion (20/~1~ of each m m # e (78.1101 ',,,as electrophore,,ed on an SDS-p~lyac~'lamide gel. The 34-kDa protem band i:, indicaled by an

asterisk,

'~'3! O I"

~,.m u .- 2

O

U m <[

B

kDa

68 ~"

1,3

29 2z;

, ! t 1' I I' "

M

IC 20 30 40 50 60 70

FRACTION

MW32 34 36 38 40 42 L./.,46 /.8

l ip 4,

0 4

0,2 K.

Fig. 3 tlydrox2,apatite chromatography ~f th,." priming enzyme. Panel A: Iraclton Nf, v, as snblected to chromalogJaphy t,n hydro~'apalJte as de,,cribcd in the text. Priming activity ',',a, measured as described

in Mdtcdals and Method,~ and in the legerd Io Fig. 1. Panel B: a portiun {5{} ,~11 of each ,,~'r:~-i~ !:~ ' ! ' ,~!:¢~re,.ed on an

The priming enzyme in the crude permeable cell extract was purified about 1 200-fold at the Sephadex G-Ill0 purification step {Table I). Although the specific activity and enzyme stability were reduced by further pt~rification using hydroxyapatite, the enzyme prepara- ti.~ became physically homogeneous, migrating as a single 34-kDa protein band in SDS-PAGE (Fig. 4). The intensity of the CBB-stained 34-kDa protein band was the lowest in the N: fraction, suggesting that the 0.2 M KPi extract contained both a phosphocellulose- absorbable and a non-absorbable form of the 34-kDa protein (Fig. 41, The adsorbable 34-kDa protein was the priming enzyme, The DNA pol vmerase priming activity of the 34-kDa protein on bleomycin-damaged or acid-de,-~urinated DNA was also detected by blotting (activity blotting) on bleomyein-damaged or acid-depurinated DNA-fixed membranes or by measuring priming activity using renatured protein extracted from gel slices [12] (data not shown).

61

TABLE 1

~ r i f i ( a l , m o f tile priming enzyme t.4P/:,Y , le . 'h , . ,e l for lgp!l, tr DP,24 ~ mhe~t~ ~r .m mtar, c a~ ~n'~ ~ur¢ . m a I{~R ( l i t " I t ~ c/t, "

Fraction 1 .t~! Total p, liming ,~cll~'it} ~DC~.IIIC aCti~ It'. bruit,, ' rngl

(me} (units~ 10 2} BI.M{ + ) / ( - 1 ~"

Crude extract 1210 4.25 1.4 0.3g 0.1~,~ l st phosphocellulose l 15 1.2~ 3 .[} 1. t 0.74 DEAE-cellnlose 59.~ 0,~67 24 1.4 IJ~h 2nd phosph{~ellufilse 9.60 I .l~ 7.4 ] 1 Ill Sept ,dex G-100 2.3 {} 2.0 0 14 12'~ I I s ssDNA cellulose n.d. d I .M I 7 n.d n d. Hydrox'/apatite 11.3116 0.102 Ib 33 32

a The priming enzsme ~APEX nuclea.~) was purified ~ the procedure de~ribed in Materials and Metba}ds. t, The priming activily for DNA polymera.,.e # was measured as described in Materials and Methods, I~LMI + ) and t - P indicate the activities

measured in the presence and absence of bl¢omycin, respectively. BLMI + )/~ -- I indicates the aetwit!, ratio or the degree of Neomycin dependency of the ao iv ib .

r BLM~ + ) - ( - ) indicates bleomycin-der~ndcn! OCtB Ity in which the actr,'ily ,neasured in the ab,.ence of Neomycin ~as ,~abtracted kom the activity measured in the presence of bleom)cin

a rid.. not determined Protein content in fractions eluted from ~,DNA edlnlo,.e ~,as nol d¢lermmed becau~ the elation baiter contained ~}vine serum albumin.

Characterization of the repair enzyme The purified enzyme showed DNA polymerase

priming activity on mierococcal nuclease-treated DNA. Neomycin-treated DNA and acid-depurinated DNA, similarly to that of cxonuclease 111 as suggested previously using the partially purified enzyme (Table I1} [9].

, .o

oO on o.O .o oo o*.e

e

88}" ~ ,

43 ~- ~

291~ -=~ ~ ----" -- 1--

24 )" - - . . . . '

., l Fig. 4. SDS-pol~.acqtamide gel electrophorests ol fractions c.b~amed in purification steps of the priming ea t 'me /.APEX nuclea~¢~ for repa~r DNA s)mhesis. Samples gere electrophote,,ed ~rn an SDS- pobacr~lamide gel. The arr~tunt of protein hmded per lane ~a- as folk~ws: the crude extract, 87 tag. the 1st pht~p-bocdluk~e fr~c~cm. 60 ~g: the DEAE~ellulose fraction28 ,ag~ the 2rid phos~:,celtuk.~*: fraction, 6a #g; the Scphadex G-100 fr~l ton. 14 p,g: the hydmr<~

apatlte fTactlon. 062 tag

As expected by its priming activity for DNA repair on Neomycin-, peplomycin- or X-ray-induced single- strand breaks (]'able il)[9,11,26], the enzyme showed 3'-5' exonuclea~ (mononucleolytic) activity on duplex DNA, like exonuclcase II1 {'Fable III. Fig. 5). The exonuclease activity of the enzyme on bleomycin- treated DNA measured under the present conditions was 50 nmol of nucleotide released/rag protein per min. The exonuclea~ activity depended on Me:" ions (data not sho~n). The enzyme also showed nicking activity on DNA having AP sites but gas inactive towards native DNA (Fig. 6} [12]. The nicking activity of the enzyme was estimated to be about 20(} ~mol of

1ABLL It

Tcmplaf{..pr~mer ,~c Itr mr~ f . r 11~,2,1 pcd),rterasc fl

prelrt atrff axth pr* tn~ , l,t~2~mc$ *

oj nuMif ied 0 % ,i

Pruli'catmt-nt ~1~ Pr*ming cn/'~mc !~HldTMP c;~!f t~)-mu~ DNA incorf'n!t ated

! pmol

Non-*reat*d ~me [I ! ? c~onude a~,c Ill ~' t}~i

{~ea!ed e~.onudea~ Ill 1.4

APEX nuc!ea~¢ 1.2

Bleom~cmqrea|ed none {}18 exonudeast l i t 5.2 APEX n u d e a ~ ?.b

]empl~t~,pnmcl acH',iI) '~.as measured as de~Tribcd in Materia|~

LJ dl urn| of ¢~om~ckray._- Ill was added v_, 60 ,al of '~he rea,cti(m mL~tur¢

• ',~J ng of p~r~f~ed rr~ms{ APEX nudease lemming enzyme} ~a$ added to 6{J .=l o{ the reactron mixture

62

ORIGIN dTTP dTDP dTMP ¢ ¢

I O

~2

0 2 /,

MIGRATION (cm)

iv Fig. 5. Assay of 3 ' -5 ' e.:onuclease activity of purified mouse A P E X nuclease. 3 ' -5 ' exonucte~rse acti,,ilY of the enzyme (fraction N 7) v.as measured using alternate ¢opolymer l~ly(dA-dT)'lX~b4dA-dTl 3'- end-labeled ~ith [3H]dTMP as described in Materials and Methods The purified. 3'-end-labeled copolymers were incubated at 37°C for ~a~'ing limes v.ith the priming enzyme in Triton-buffer B. Nu- cleotides released h) exonuclct,lylic digestion ~ere analyzed by polyethylene /mine-cellulosc thin l*lyer chromatography as previously described [2S] :. 311 min: • e, 60 rain: ~ ~.

12tl rain.

a c i d - d e p u r i n a t e d p U C I 8 D N A n i c k e d / m g p ro t e in p e r

21t rain by m e a s u r i n g t he a m o u n t o f e n z y m e r e q u i r e d to

conve r t o n e - h a l f o f the supe rco i l ed a c i d - d e p u r i n a t e d

p U C I 8 D N A to n icked , o p e n c i rcu la r D N A u n d e r t he

~,say condi t ions de sc r i bed in Ma :e r i a l s a n d M e t h o d s .

The A P e n d o n u c l e a s c act ivi ty was a lso d e p e n d e n t on

Mg 2. ions and d iva len t cat ions , sach as M n 2" and

Lo- , can substitute for Mg 2~ ion. The enzyme l~s- sessed DNA 3' phosphatase activity (46 nmol of Pi relcased/~;ig protein pcr 3',) min) (i:g. 71.

"rhis priming enzyme is denoted as APEX nuclease becau~ of its major characteristics. AP cndonucleasc and exonuclease activities.

The amino acid composttion and the atnino-terminat and internal amino acid seqttence~ of AP;~X nmh'as,'

The results of amino acid anal~si~ of the purified mouse APEX nueleasc arc shown in Table IV. Auto-

FABLE Ill

Exonuch,as~' aeli~itie~ o f t:to~ :cast III and p:v,fied memse APE.,," lltl¢ h'asC

Acid-dep~inated ONA Native DNA

f II I I 2 3 4 5 6 7 8 910111213141511617

OCl=.

SC

Fig~ ft. Assay for the AP endonuclease activity of purified mouse A P E X nuclease. The AP endonuclease activity of the enzyme (fraction N 71 was measured as described in Materials and Methods, with the following modifications: Lanes 1 to 13, a¢id-depurinaled pUCI8 DNA: lanes 14 to 17. native pUCI8 DNA. Amounts of enzyme protein in the assa~, mixtures (12 V.I) were zero in the samples electrophore~d in lanes I and 14:3 !0g in lane 2:30 pg in lanes 3, 7. 11 and 15:3t10 pg in lanes 4. 8, 12 and 16: and 3(~10 pg in lanes 5. 6. 9. l(t. 13 and 17. Mg 2" ion was omitted in the assay mixture electr*~phoresed in lane 6 and Mg 2" was replaced with Mn2" ion in lanes 7-q. Zn 2" in lane 10 and Co:" in lanes 11-13. Abbrevia- tions: PC. nicked, open circular pUCI8 DNA: St', supctcoiled

pU('18 DNA.

matcd Edman degradation and gas-phase amino acid sequencing was a l ~ performed and the following amino-terminal amino acid sequence was obtained: N-Pro-Lys-Arg-Gly-Lys-Lys-Ala-Ala-Ala-Asp-Asp-Gly- Glu-Glu-Pro-Lys-S=r-?-?-?-Thr. Treatment of APEX nuclease with formic acid that is known to hydrolyze peptide bonds between aspartate and proline cleaved the enzyme into a 30-kDa fragment and a 4-kDa fragment. The two fragments were purified by SDS-PAGE. A partial amino acid sequence obtained by analyzing the largc fragment was as follows: (Asp)-Pro-?-Asp- GIn-Lys-Th r-Se r- Pro-Ser-Gly-Lys-Sc r-Ala-Thr-Leu- Lys-lle-?-Ser-?-Asn-Val-?-Gly-Leu-?-?-?-lle-Lys-Lys-?- ?-Lcu. The amino-terminal amino acid sequence of this enzyme is very similar to that of the calf thymus AP endonueleasc reported by Henner et al. [19] (Fig. 8), altbot.gh the amino acid composition of mouse APEX nuele~se is significantly different from that of the calf thymu, AP endonuclease "~,ith respect to the contents of alaninc, glycine, lysinc, phcnylalanine and tyr(~ine [191.

(pritr2ll?g e/lztprlt,~ a

Pretreatment i~f Pr ~ol tfl n.=cleotidc relca,,,:d/t-~t rain [ ~ HklTMP-labeled /:' ('~dt DNA ,,x~t~d¢ ,,c 11l APEX nuclease

t().{) | Unll I (0.2 U g prl~tein)

Non-tr¢~led 94 56 Bleumycin-treated 40 2It Micr~x:t~cal nucleasc-trcated 91 2S DNasc I-lleatcd 1¢~6 ~,7

" Preparation of hlcc~m~,cin-, micr~.occ,iI nl=cle~,~c or I)N;~c I Ir~';tlcd : l t~ d',,,:k'd [)NA ,~ntl a~,,,;~, to~ ¢x~mudeJ,e ~ = ~ . ~ l l ~ . ' . , v, erc corldnoud as described m M~le=hds and Mcfl~ods

(,3

]c~ ~ .*.~.,? f,,r Ih r DN . "~, I:~;e-ph;mL;m,u ;,L'lmlmkk ,~1 ImurmbmL r~.mu~" 6 i~m~d ~ a l l a¢(i~i(~' hlotl i~l~'. I ¢ ¢ h r l ~ u ¢ ~1Z].

~uleriul~ and ~Jclhrd~ ,~,PF)C ~.c l ,u~. IIT~cht~n h- 113 n[Zl ' ~ klUnl3r~4 l~l---~-~]. ' r h : prcVi<lu~l~" dlJXL~'ih~d mxmmzEi~ lT,

~)in I~l~zl of) ' r iu~n.h.~er l~ .The Tele; l~L]J: : l '~m"~a~iL 'F~;~ UX4JIILICld~.'I~ ¢1 I J l l c ~ p h a h l ~ ~ ,c l i¢ i r~ . , ir~ con t ras t Do

"L'~BLE I~' cn r61 iTuc lca~ (~-~a~ I l l a n d 3 ' . A P ~ n d L ~ L L C ~ . ~ (c!a~,~ I

. ~ , i i I ~ I ¢ -.~.~F.Ir*~'n~ .%: ~ clca,.= ( .4P. 'v ' I c n z ) m ¢ ) ha~ D)%~. 3 ' ~cpaJr dl¢*]¢r;4~c

G b T i ~ S~.l ch~" ]3?~t ~, . rod pscdlc'tc£L : ~m im+ acid ~qUeT~L~¢ I'O[' lh¢ 1 Ik l i lbn¢ ~'.~

I¢l l l ' ine ~:.:, , . 'RLodin~ D N , ~, Cl )J~r ;u~lL 'd~" I V ¢11 ~ rr~'l [!¢.~-~...~5J. I Iy~ne 2~..; ~,)l fHr a~ ~'L" ]~nL:'~I t he hi(DJ~r~o C':prYFI¢~t Hmmn[~ ~LC3d )'l~'rh!Ilnlle ! 'b ~cc~ucn¢¢~ 411 L'Uk~LryLktic ..s.p CDc~Jl)tlclcasC'~ ~ r c r) l¢ Pl~li¢." I . , . : ~.CSS[ ..)~.%:d . :n .~Tnc [.'~4,3~,J. t h= ? red i c [ cd , ~ n ¢ prc~] I

")l,dr61r.;n~ )IRI ~ : l )~r Lhym:J~ i 'ke ¢lmduml)clcak~" Ll~)J r h ~ , pPIrLib~L a~;~l¢) T)r<hane ' ~ E i~L ~ . U ¢ ~ , : S Or" A P E X . ~ucmc~lc q~, n o %I~T[I[B--JITIL

~!'.~1~'~11~. :~JiP¢ .l~d I~l~lr~h~n I~:" Jl~" f:uI L]'.'IClm!II='~ ~ ! '~h

L~,=Lummi[mJtio¢l ;i,. ld comDar isc)r l ~ [ t l~e oori lpmcz¢

64

functional relationships between the various species of mammalian repair enzymes including AP endonucleasc. We are now attempting eDNA cloning for APEX nuclease using probes of oligonucleotides deduced from the determined partial amino acid sequences. Such an approach is also important for studying repair deft- cient-inherited diseases.

Acknowledgements

We thank Mr. T. Nakamura and Ms. T. Yasul lor their technical assistance and thank Nippon Kayaku for providing copper-free bleomycin A,. This work was supported in part by a Grant-in-Aid for Scientific Re- search from the Ministry' of Education, Science and Cultured of Japan.

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a mouse dna repair enzyme (apex nuclease) having exonuclease and apurinic/apyrimidinic endonuclease...

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