BIOLOGIA PLANTARUM (PRAHA)

19 (~i) : $46--352, 1977

Isolation from Barley Embryo of Endonuclease Specific for Apurinic Sites in DNA

J. V ~ i ~ s x ~ , Jr~x~-A ~WCHUmVA and J. ~ATAW,

Insti tute of Experimental Botany, Czechoslovak Academy of Scienvea, Praha*

Abstract, Th~ r activity speeifi~ for apurimc sites ~n DNA was db~eeted in barley embryos. The cnzym~ w ~ partially purified. I t reveals high activity on partially deputin~ted DNA bu~ low or nil e~tivity on in~ac~ az~d allry~+~e~ D:NA. The method tree4 for the detection of etmyme ~ i v l t y w ~ based o n the changes in the sedimentation velocity of sabstrate ~ N A ha n~utra~ aucrose gradients with 80 % formamid~,

Er~lonuc[e~se speeis for apurinie and apyrimidinie sites in DNA was isolated from several organisms (VERL~ et aL 1973, T o ~ I N 1974, LJvl~c- QWST and LI~pa~L 1974, KUK~LV.r~ el al. I976) including plants (TBxBo- nE^u ~nd VERLY 1976, V~RLY et al. 1973). I t is supposed that this enzyme plays an important role in the repair of mentioned sites in I)NA which occur either spontaneously or are induced by mutagens (el. VE~LY et al. 1973, LJUI~QQ~IST and LIZ(nA~L 1974). l~ecently this enzyme was isolated from barley leaves and its specificity was determined (SvacHunov/ et al. 1977). lqere we show that a similar enzyme is also present in cells of barley embryos. If was already demonstrated that in these cells an excision like repair of DNA single strand breaks induced by alkylating agents (V~Lv.Mi~s~:# et ~,I. 1972, 1973) including repair synthesis (VELEMi~SK% et at. 197.7) takes place. I t is higtdy probable that the endonuclease which attacks apurinic sites and produces single stra~nd breaks participates in t h h repair process.

Material and Methods

Barley seeds Hordeum vulgate (cv. Dvoran) were soaked in wafer and then the embryos were separated from the endosperm. Free embryos were used for the enzyme isolation according to the schedule described by PA- QVETT~ et al. (1972) originally for Esvherichia coli. In short the method included homogenization of embryos, precipitation of nucleic acids with streptomycin, ammonium sulph&t~ fractionatiou, D E A E cellulose chroma$o- graphy and phosphocellulose chromatography. The details are given below (see Results). All operation were carried out at 0--4 ~

Receive, d M a ~ h 7, 1977 *Addrr Flemlngovo ns 2, 160 00 Prsha 6, Czvohozlovaki~.

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E N D O N U C L E A S E S P E C I F I C F O R A P U R I N I C S I T E S I N DI~A 347

DNA used as substrate for enzyme action was isolated from barley embryos according to M/minim (1961). Sedimentation coefficient s~0~ of this DNA as measured in analytical ultracentrifuge (Beckman Model E) varied usually around 26--28 S. DNA labelled with thymidine-(3tt) was isolated from Bacilhts subtilis B/3 thy - according to the same method (MAIt~tVR 1961). Its radioactivity was roughly 108 cpm ~g-1. This DNA-(ZH) was built into the polyacryiamide gel as described by MELGAI~ and GOLDTat- WAI~ (i968) and TO.~ILrN (1974). Alkylated DNA was prepared by incubating the intact barley or B. subtiIis DNA with 50 mM MMS (final concentration) at 37 ~ for 30 min. Depurination was carried out by heating of alkylated DNA at 50 ~ for 6 h.

Protein fractions or enzyme preparations were tested as follows:

1. Suerose~Formamide Gradient CentrllttgaUon

The protein samples were incubated with unlabelled barley intact, alkyl- ated or depurinated DNA (1 : 1 vol/vol) for 60 rain at 37 ~ with addition of ggCl~ up to 5 mM. The concentration of DNA was usually 2.5 ~g ml -I. The reaction was stopped by adding 2 vol. of cold 96~ ethanol and the precipitate was dissolved in 0.15 M NaCI + 0.015 M sodium citrate (SSC). The control was incubated with buffer only. Enzyme activity was measured by the decrease in the size of DNA, comparing the sedimentation profiles of DNA incubated with the isolated protein to the control DNA. Sedimentation profiles were obtained using Spinco L2-65B centrifuge (Beckman), rotors SW 27.1 or SW 56Ti. After the centrifugation the tubes were punctured and the absorbance of effluent was continuously recorded in differential UV-ana- lyser. Enzyme activity was expressed semiquantitatively as a difference between the position of maxima on the sedimentation profiles of digested and control DNA. To detect single strand breaks, neutral 1--15% (w/v) sucrose gradients were made up in 80 ~/o formamide-0.1 M Tris-tICl buffer of pH 7.4. In this medium denaturation of DNA takes place as evident from the determination of hyperchromieity of aliquots after centrifugation. No spontaneous eleavage of DNA at the apurinic sites occurs in this medium (Ts'o et al. 1962, GA~'~Izr and YIErmIz~G I972). The rehability of the for- mamide method was verified by comparison with usual alkaline sucrose gradient (5--20% sucrose w/v in 0.9 M NaCI, 1 mM EDTA and 0.3 M NaOH

:Fig. 1).

2. Teatinff on DNA.,Gel SubtJtrate

The protein samples were added to the mixture of 0.25 M Tris-HCI, 0.03 M MgCI~, pH 7.5 and DNA-gel suspension (about 1 ~g DNA) and incubated at 37 ~ for 60 min. The mixture was then centrifuged at 5000 • for 3 rain and the radioactivity of the supernatants measured in a scintillation counter.

Results

Embryos isolated from 150g of seeds were homogenized in 25 ml of Honda buffer (0.03 M Tris-HC1, pH 7.8 with 0.25 M sucrose, 2.5 % Dextran 40, 5 ~/o Ficoil, 5 % polyvinyl pyrrolidon and 0.1 M MgCle), and the slurry

Abbrevlaaxm trued: MMS = m e t h y l methan~su lpko~aLe .

348 J. VELEMTNSK~ ET AL.

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20 40 80 80 tOO ~ t o p ~ of gradlent length

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Fig. 1. Model exper inmnt demons t r a t i ng sui tabi l i ty of the sucrose . formamlde grad ien t r fugat~on for the es t imat ion of the apurinic D N A size. A. Alkaline sucrose gradient , ~W 27.1 rotor, 25 000 rpm, IS h, t~ ~C. B. Neutral suerose.formamide gradient (I--15 9/0 sucrose ~']v, in 80 % formam/de) , SW 56 Ti rotor , 55 000 rpm, 6 h , 25 ~C. Curve No. 1 = in tact Dh*A (calf thymus) ; 2 ~ apvxinio D N A (calf t h y m u s D N A alkylat~d wi th 0.25 mM ?~IM~q for 4 h a n d cl~purinated s t 60 ~ for 6 h); 3 ~ apurinie D N A -{- Zx'aOH (2 volumes of the D N A sample dissolved in SSC were mixed wi th 1 volume of l N N a O H , incuba ted a t 37 ~ for 30 m~n, t-hen neut ra l ized wi th 1 vol 1 N HC1 and layered on suoross- formamide gradient) . Fig. 2. Enzyme activity of barley embryo extracts after different steps of purification, measured in suorose- formsmide gradients (SW 56 Ti rotor , 55 000 rpm, 6 h , 25 ~ A. 1 = apurinio D N A -~- -{- I~'aOH; 2 ~ apurinio D N A -{- rough ex t rac t ; 3 ~ in tac t D!qA .~ rough ex t rac t ; 4 = apurinic Dlh'A -{- H o n d a buffer; 5 ~ in tac t D N A -}- H o n d a buffer. B. 1 = ~puriino D N A -{- rough ex t rac t ; 2 = apurinle D N A -k extrac~ a f te r s t r ep tomyc in s u i p h a ~ a n d (,WHa)zSO4; 3 = spur /h ie DlqA -{- e x t r a c t af ter s t r ep tomyc in sulphate ; 4 ~-- apurinio D N A + H o n d a buffer.

was centrifuged at 10 000 x r for 20 rain. The supernatant (called further as rough extract or Fraction I) was mixed with streptomycin sulphate (1.6 % in 0.05 M Tris-HC1, pH 8) i : 1 v/v to precipitate nucleic acids and the mixture centrifuged at 10 000 • for 15 rain. The supernatant designated as Fraction IX was precipitated with ammonium sulphate. Proteins precipitating between 45--80 ~/o saturat ion were collected by centrifugation at 10 000 • for 18 rain. The sediment was dissolved in I0 ml of 0.05 1~ Trls-HC1 buffer pH 7.8, with 10-4 M EDTA and ammonium sulphate was removed by dialysis against the same buffer (Fr~ction III). All fractions described up to now were tested on the ability to nick both apurinic and intact DNA (Fig, 2B). As demonstrated in Fig. 2A, the rough extract (Fraction I) degraded the intact and the apurinie DNA to about the same extent as did the incubation of apurinic DNA with NaOH.

ENDONUCLEABE SPECIFIC FOR APURINIC SITES IN DNA 349

Fraction I I I was chromatographed on the DEAE cellulose column (2 • 7 era) by gradient clution with 200 ml of 0.05 M Tris-HC1 buffer pH 7.8 and 200 ml of the same buffer with 0.25 M NaC1. Ten ml fractions were collected and from each of 8 successive fractions 3 ml were pooled and concentrated on Diafio membranes under the pressure of nitrogen. In this way 5 groups were formed from original 40 fractions and these groups were tested on their activity to apurinie DNA {Table 1, left upper part). The highest activity, as measured by the decrease of the sedimentation rate of apurirdc DNA, was present in fractions I I U I - - 8 and III /9--16. The ac t iv i ty of further groups was so low tha t a fur ther testing was omitted. Original fractions 1--16 were then tested individually on apurinic DNA. The results summarized in Table 1 (right upper part) indicate tha t all fractions starting with number 5 revealed sufficient activity. These fractions (5--16) were therefore pooled, 4 times concentrated and tested On intact, a lkylated and

pNlirinic DNA. This preparation (Fraction IV) was fairly specific for apurinic A; the activity in intact or a]kylated DNA was 17 or 6.5 % respectively

~s compared with apurinic acid (100 %) -- (cf. Table I -- lower part). Fraction IV was dialyzed against 0.04 M sodium phosphate buffer pH 6.5,

layered on phosphocellulose column (2 • 7 cm) and eluted with 2 X200 ml of 0.04 M sodium phosphate buffer pH 6.5 with 0 - 0 . 3 M NaC1. Aliquots of I 0 ml fractions were tested on their act ivi ty to apurinic SH labelled Bacillu, a.sbtiH, DNA (Fig. 3). The selected fractions whichrevea led higher act ivi ty on this substrate were further tested on barley apurinio DNA (analyzed in

Activity of f r6ot io~ collected after DEAE-vellulo~0 ch~mAt~gr~phy, me~ntred Aooordlag to t i~ docreas~ ~ m ~ m t ~ o n velocity of variou~ smbs~ .~ DI~As, ~ compared to oont4-~l D~A, i~

Buc~osv gr~dien~ with 80 % formamide. Tim dat~ in the t~blo roimment ~ho difference between m ~ m ~ of both sedimentation ea rv~ , exIJ*~ed in % of total gradient length. Centrifug~tion w ~ carried ou~ in the rotor SW 27.1 s t 26 000 rpm, 20 h, 25 aO

F r ~ t i o ~ activity on Fraction Np, 8otivity on apurmic DNA Apurir~ DNA

I . l . I f l--s 12.~ I I I ] l 2.4 2 3.8

rrf]9---16 12,2 5 4.8 6 9.3

I I I /17- -24 4.4 7 6.9 $ S.7

YI'/]25---32 4.8 9 6.1 10 7,4

III~33~ i~).@ II 15-7 13 8,5 15 6.8

Activity on le~aotiona intact DNA alkylated DNA apttnnie DlqA

~ 1 6 (eoucentr.) 3.3 1,2 19,1

350 J. VELEM][NSK'~ ET AL.

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:Fig. 3. Enzyme ae t iv i~ os fra~tione collected after phosphocellula~ c ~ o . matography, measured a e ~ d L ~ Co 3H radioactivity released from apurmic DNA SH - - gel substrate.

neutra l sucrose-formamide grad ien ts )and intact DNA (analyzed in alkaline and neutral sucrose gradients). All fractions tested were highly active o n apurinic DNA (Table 2). High activity of fractions IV/5, 17, 31, 34, 37, 39 on intact DNA and the inability of fractions IV/8, 14, 21 and 24 to induce

TA~L~ 2 Aetivir of fractions collected after phosphoc~lI~lose ehromAtegraphy, me~aumd aecordlng to the decreased sedimentation velocity of auhstrat~ DNA, as ea~npared to control DNA, iu sucrose gradients. The data in the t~blo represent the difference b~tween maxima of both sedimentation curves, expressed in % of total gradient length . . . . Centrifugation of alkaline and neutral sucrose graditmts was carried out at 5 ~C either, in the rotor SW 27.1 a t 24 000 rpm 18 h or in, SW 56 Ti a t 56 000 rpm 2 h. Oontrifugation of neutral sucrose gradient with 80 % formamide was carried out in tho rotor SW 66 Ti, at 55 000 rpm 6 h, 25 ~

Apurinic Dlq'A I n ~ c t DNA Fraction No. sucrose grad. sucrose gradlenia

with formamido alkaline neutral

rv]5 30,8 10.6 10.2

8 24.7 - -0 .9 - -1 .0

14 30.0 --1.3 - -1 ,0

17 19.2 11.5 24.2

21 25.9 --1.1 - -0 .9

24 9.0 - -3 .8 - -2 ,3

31 12.2 6.0 13.5

34 21.5 12.,5 10.3

37 15,1 33.3 15.6

39 20.0 17.5 4.4

ENDOI~CLEASE SPEC:IFIC I~0R APU-ItlNI0 SITES IN DNA 351

single-and double strand breaks in intact DNA indicate that after the phosphocellulose chroma*ography at least two proteins were eluted which both revealed the specific activity to apurinic DNA -- one containing the fractions 8--14 (peak B, Fig. 3) and the second ~he fractions 21--24 (peak D, Fig. 3). Obher peaks comprising e.g. the fractions 5, 17, 31--39 (i.e. peaks A, C, E, F, G in Fig. 3) contain nonspecific nucleuses, producing single- and double strand breaks in both i n ~ and depurinated DNA.

~7~ 7 Disenssion

In our previous work (Sv~c~vL0vk et el. 1 9 7 7 ) isolation of specific endo- nucleuses from barley leaves was described. On the hydroxy,pati te columns two samples were resolved, the first being active on both intact and de- purinated DNA, the second degrading specifically apurmic acid. In the present work two peaks were also obtained after the chromatography on phesphoeellulose both specifically active to the depurinated DNA. Further comparison of samples from embryos and from leaves was not possible due to the low amount of enzyme isolated from embryos,

High degree of speoifici~,y ~o depurinated DNA, becoming apparen~ after nonspeeifie endonucleases were removed by the chromatography on DEAE cellulose and phosphocellulose, a]lows to conclude that the endonuclease specific for apurinic sites in DNA is present and active in that stage of em- bryonic cells in which *he repair of DNA single strand breaks and repair synthesis, induced by alkylating agents, were detected (V~Lz~i~SK$ et al. 1972, 1973, 1977).

In the present work a new method suitable for the detection of enzyme activity was developed. It seems to be suitable in those cases where the subs~rate DNA is not or cannot be labelled.

A~nowIedl lemeuls

We wish ~o thank Dr. N. V. Tomflin (Institute of Cytology, Aoademy of Sciences USSR, Leningrad) for learning us the method w~th DNA-poly- acrylamide gel and to Drs. J. Dosko~il and V. Pa~es (Institute of Mole- cular Gcnetiscs, ~SAV, Praha) for the constructive criticism of the manu- script. I ~ Beferenees

GAUDIL~ D, , Y]~IDL~ffO, K. L. : ~lete LIBO Of fo/~lT~l~dO gradients to dlsthag~sh be tween alkali labile regions and slngle.strand breaks in D N A . - Biochem. biophys, l~s -Commma. 47 : 1396 to 1401, 1972.

:KuHr~t,~r, U., Pm~tOE~l', E, E,~ Llr~r, 8.: An alt~lw~1 ~,purinie DNA endon~clesse actdvlty in grOUp A ar~I g roup 23 Xewodr :~iffrcicrdoa~ ilbroblasts. - - ]?roe, ~ t . Acad , 8ei, U S A 75 : 1169--1173, 1976.

L~V~OQVIST, S., LIl~mam., T.: A nmrrmmlian endonu~leaso specific for apurmis site~ in double. s t randed deoxyribonucleic acid. - - J . biol. Chem. 249 : 1530~-1535, 1974.

Mx~mm, J . : A procedure for isolation of deoxyribonucleic acid from microorgsaaismus. - - J . mot. Biol. $ : 208--218, 1961.

M.~T.OA~, E., GOLnXmwArr, D. A. : Deoxyribonucleic acid nucleuses. I. The use of a new method to observo the kinetics of deoxyriboaucldc acid degradat ion by deoxyribonuelet~so I , deoxy- r l b o n u e t ~ I I , a~d Eaeherivhia ool~ ozxdonuel~,~o I. -- J . biol. Chem. -~ ; 4401--4408, 1968,

PAqm~a:g, Y+. C1~.xlcm, P., Vm~Y, W. G,: Properties of the er~tonucle~so for depurinatod D2ffA from E~hef/cAia coll. - - Caned. ~. Biookorn. ~ : 1199--1~09, 1972.

3 5 2 J . VELEMINSK~" ET AL.

gvxcnUl_OVg, J. , ~ATAVA, J. , V~LZ~rfXSK~-, J . : A barley oadonueloase specific for Apur/nio D17A: Isolation mad part ial eb~raetorisation. - - Eurep. J . Biochom. (Submit ted for publication.)

Tm~ODEAU, L., V ~ x ' , W. O. : Endonucleaso for apurimo sites in p l a n t s . - FEBS Lott . r : 183 to 185, 1976.

ToMx~r, lg. V. : [On the p r e ~ n c e of an e n d o n u c l ~ e acting on LrV - - irradiated and depurinated DNA in M~rococcua Iyeod~ih..ti~'u~ cells]. I n R u s s . - 1~Iol. Biol. 8 : 557--~568, 1974.

Ts 'o , P. O. P., HEnri,:AMP, K,, Sx~Dls~, CrL: Secondary structure of nucleic a~ids in organic solvents. I L OptieM properties of nuelootides and nucleic acids. - - Biochim. biophys. Aeta 55 : 584--600, 1962.

V~I~ri~rsg,2, J. , ZAI~I~Af~IL, S., Glcm~E~, T.: Repair of single.strand breaks in I)NA and re~owr? of induced mutagcnie effects during the storage of ethyi-mebhano-su.tphonato-troaeod barley seeds. - - Mutat ion Roe. 14 : 259--261, 1972.

V ~ r ~ i x s x ~ , J . , Z ~ n ~ r ~ , S., Poxomn~, V., Grcmc~R, T., ~vxc~tyr~ov~, $.: Repadr of single. -strand breaks aztd fat~ of N-7.methylqua~tine m DNA during b1~o recovery from genetie~ dalnage induced by :N.methyl-lq-nitrosourea in barley seeds. - - Muta t ion Roe. 17 : 49---.~8, 1973.

V l ~ I . & ' g ~ , J . , ZADRA~,Ir~, S., POgORN~, V., GIOHlCEI':, T.: DNA repair synthesi:~ s t imula t~l by mutagenie N-Methyl-lg.nitrosouroa in barley seeds and free embryos. - - Mutat ion Res. {in press}.

VE~zLY, W. O., PAqtrs'I~E, Y., TKIBODI~AU, L,: Nuclease for DNA apurinie sites may b e / n v o l w d in the m a i n t e n ~ c e of DNA in normal oolls, ~ Natura 244 : 6 7 - - 6 9 , 1973.

BOOK R E V I E W

K g ~ m c g , R. E. , ~ N D , B.: Phytochrome and Plant Growth. Studies in Biology" 17o 68 . - - E d w a r d Arnold Ltd., London 1976, pp. 68. s 1.50.

The booklet ia a competent guido in the Reld of phytoehrome.mediatod p h e t o m o r p h o g e n e ~ . The importance of this probl~m has increased in z~c~nt times, due to the dis~ov~y of a close link between photoperiodm phenomena and emdogenous rhythms.

The following problems are dealt with in individual chapter~: some basle terms, such a~ rod/far-rod roxnorsibility, action spectra and absorpt ion spectra are explained in introduction.

Five chapters are devoted to phytoehrome detection and isolation, properties of phytoehrome, phybochrome-controlled raspormes, mode of action of phytoehroma and suggestiona for praeOeal exercises. The authors contr ibuted a great, deal to our knowledge of bioehemistry of phyt~ohrome and its phys/ologioal action. They are competent to appreciate reliably Eho methods and give suggestions for practical works. This part icular t ra i t makes the toe, ted material more unders tandable and palatable.

The publication brings highly instruct ive figures and tables and a list of selected literature. Although the book is assigned be undergraduates in biology, i t can be usofu] f~ everybody who

wants to keep pace wi th the progress in this field. I t provides basic information and points to eu r~n~ ideas,

J . K ~ x v ~ (Pea/m)