Eur. J. Biochem. 87,215-220 (1978)

A Barley Endonuclease Specific for Apurinic DNA Isolation and Partial Characterization

Jifina SVACHULOVA, Jan SATAVA, and Jifi VELEMiNSKY

Institute of Experimental Botany, Czechoslovak Academy of Sciences, Praha

(Received January 3/November 28, 1977)

An endonuclease specific for depurinated native DNA was isolated and partially purified from extracts of barley leaves. The procedure included streptomycin sulphate precipitation, ammonium sulphate fractionation, phosphocellulose, hydroxyapatite and Sephadex G-150 chromatography. Purity of the resulting enzyme was determined by gel electrophoresis and gel chromatography and specificity by testing the activity on intact and depurinated bacterial DNAs. At lower concentrations, the enzyme is specific for DNA containing apurinic sites. At higher concentrations, however, it degrades DNA in a non-specific manner. The nuclease has a pH optimum at 7.6, and a molecular weight of about 18 000.

Endonuclease activities which introduce single- strand breaks into native DNA containing apurinic sites have been found in extracts of bacteria [I -41, mammalian tissues [5], human cells [6,7] and also higher plants [8-lo]. There is good evidence to support the assumption that at least some of these enzymes, called apurases, play a role in the repair of apurinic sites in vivo. Mutants of Escherichia coli with low levels in apurase are sensitive to methyl methanesulphonate [I 1,141. In human fibroblasts derived from patients with xeroderma pigmentosum complementation groups A and D apurase manifested lower activity, different pH dependence and altered K, compared to the apurase of normal human fibro- blasts [7]. In cooperation with E. coli polymerase I and T4 ligase, apurase from either E. coli or Micro- coccus luteus completely repaired depurinated DNA in vitro [12,13]. The relatively high rate of spontaneous depurination of DNA in vivo is also hardly compatible with a low rate of spontaneous mutagenesis or cell lethality without assuming a repair function of apu- rases in living cells [15].

The enzyme has been purified from E. coli [16,17], rat liver [18], calf thymus [I91 and Phaseolus [20]. The barley apurases have previously been purified from barley embryos [21]. In this paper, their isolation

Abbreviations. NaCl/Cit, 0.15 M NaCl in 0.015 M trisodium citrate, pH 7 ; Hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

Enzymes. Deoxyribonuclease I (EC 3.1.4.5); apurinic-DNA- specific endodeoxyribonuclease or apurase (EC 3.1.4.-).

from barley leaves is described. Part of the data published here has already been presented previously

In barley, recovery from genetic damage [23] induced by several alkylating agents, is accompanied by the rejoining of single-strand breaks [23] and repair synthesis [24]. This communication presents additional data concerning the probable steps by which alkylated DNA may be repaired in higher plants.

[221.

MATERIALS AND METHODS

Apurase activity was assayed either according to Tomilin [4] or according to Paquette et al. [16] using the modification described by Ljungquist et al. [25]. E. coli B/3 thy- [thy~zine-~H]DNA (about lo3 counts . min-' . pg-') or E. coli [32P]DNA (about lo4 counts . min-' . pg-') were used as substrates.

In Tomilin's method 2 ml of labelled DNA (100- 250 pg/ml) were mixed with 4 ml of the solution con- taining 10 % acrylamide and 2.5 % N',N-methylene- bisacrylamide in 0.05 M Tris-HC1 (pH 7.5), 0.15 M KCl; before polymerization, 0.5 ml of 0.004% ribo- flavin solution was added. The gel with the DNA in- corporated was then homogenized in a Potter-Elveh- jem homogenizer and the suspension washed several times by centrifugation and resuspension in 0.05 M Tris-HCI (pH 7.5), 0.15 M KCI. This DNA-gel suspen- sion is referred to as intact DNA. For treated sub-

216 Barley Endonuclease Specific for DNA Apurinic Sites

strates, the DNA-gel suspension was mixed with an equal volume of 100 mM methyl methanesulphonate in the same Tris-HC1 buffer, incubated at 37 "C for 30 min and washed by centrifugation (alkylated DNA), then incubated at 50 "C for 4 h and washed again (depurin- ated DNA).

For the enzyme assay 0.3 ml of the substrate DNA- gel suspension (approximately 1 pg DNA) was pipetted into 0.5 ml of the enzyme solution plus 0.2 m10.25 M Tris-HC1 (pH 7.5), 3 mM MgC12 and the mixture was incubated at 37 "C for 60 min. The reaction was stopped by the addition of 0.1 ml 0.2 M EDTA and cooling the mixture to 0 "C. Following centrifugation at 5000 x g for 5 min, the radioactivity of the super- natant was determined.

The second method of enzyme assay was carried out as follows. Labelled DNA was mixed with an equal volume of 100 mM methyl methanesulphonate in NaCl/Cit, incubated at 37 "C for 30 min, precipitat- ed with ethanol ( l / l ) and dissolved in NaCl/Cit (alkylated DNA). Depurinated DNA was prepared by a further incubation for 4 ha t 50 "C and reprecipita- tion from ethanol. The endonuclease assay mixture contained 0.6 ml 0.1 M Hepes/KOH (pH 7.5), 3 mM MgC12, 0.1 mM dithiothreitol, 20 pg yeast tRNA, 500 pg serum albumin, 0.2 ml of the substrate DNA solution (roughly 10 pg DNA) and a limiting amount of endonuclease activity. This mixture was incubated at 37 "C for 30 min, chilled to 0 "C, and an equal volume of 0.8 M perchloric acid added. After 5 min, the mixture was centrifuged at 10000 x g for 10 min and the radioactivity in the supernatant was deter- mined.

Chemical homogeneity of the purified enzyme was tested by gel chromatography and polyacrylamide electrophoresis. 0.5 ml of the sample was applied onto a Sephadex G-150 fine column (1 x 50 cm) and eluted with NaCl/Cit. Gel electrophoresis was carried out as described by Davis [26]. The gels were either stained with Coomassie brilliant blue to locate the protein bands or cut into pieces from which the proteins were eluted with 0.3 ml of 0.25 M Tris-HC1 (pH 7.5), 0.15 M KC1 and their activity was measured as described. Molecular weight was estimated by gel chromatography in Sephadex G-350 Fine (1 x 50) and by electrophoresis in dodecylsulphate/polyacrylamide gels [27] with cytochrome c, chymotrypsinogen, lyso- zyme and deoxyribonuclease I as standards.

Protein concentrations were determined by the method of Lowry et al. [28] or spectrophotometrically as described by Waddell and Hill [29].

RESULTS

Apurases were purified from the leaves of 14-day- old barley (var. Ametyst) by a modification of the

0 2 4 6 8 Fract ion number

Fig. 1. Phosplzocellulose chr.onzutography qf fruciioii I I . Column size 4 x 40 cm, flow rate 1 ml . min-', fraction volume 100 ml, buffers see Materials and Methods. (O----O) ,4280; (O~-O) enzymic activity on intact [thyniine-3H]DNA; (m--W) enzymic activity on apurinic [ t h y m i i ~ e - ~ H ] D N A assayed according to Tomi- lin [4]

method of Ljungquist and Lindahl [19]. All opera- tions were carried out at 0-4 "C.

About 1 kg of leaves was washed with 0.3 M sucrose and homogenized in a Waring blendor in 2 vol. of 0.1 M NaCl, 0.05 M Tris-HC1 (pH 7.4), 1 mM EDTA, 0.1 M 2-mercaptoethanol. The homo- genate was stirred gently for 2 h, filtered through four layers of gauze and centrifuged at 20000 x g for 50 min. The supernatant (fraction I) was mixed with an equal volume of 1 mM EDTA (pH 7.0) and then 0.1 vol. of a 5 solution of streptomycin sulphate was added to the mixture with stirring, which was continued overnight. The nucleic acid precipitate was removed by centrifugation at 20000xg for 20 min. Solid ammonium sulphate was added to the supernatant, the pH was maintained at 7.2 by addition of NH40H and the proteins precipitating between 45% and 65% ammonium sulphate saturation were collected and suspended in 0.01 5 M potassium phosphate (pH 7.2), 1 mM EDTA, 0.01 M 2-mercdptoethanol by stirring for 2 h. After dialysis against three changes of 2 1 of the same buffer for a total of 50 h, the precipitated

J. Svachulovi, J. Satava, and J. Veleminaky 217

0 5 I0 15 20 25 30 35 40 F r a c t i o n number

material was removed by centrifuging at 7000 x g for 30 min. The supernatant (fraction 11) was applied onto a phosphocellulose column (4 x 40 cm) equi- librated with 0.015 M phosphate (pH 7.2), 1 inM EDTA, 0.01 M 2-mercaptoethanol (Fig. 1). The col- umn was washed with 1 1 of the starting buffer and the bound protein was eluted with the same buffer con- taining 1 M NaCI. The fractions containing protein (fraction 111) were collected, dialyzed against three changes of 1 10.005 M potassium phosphate (pH 7.2), 0.01 M 2-mercaptoethanol and applied onto a hy- droxyapatite column (4 x 20 cm) equilibrated with the same buffer (Fig. 2). Elution was carried out with a linear gradient of 0.015-0.4 M potassium phos- phate (pH 7.2) in the presence of 0.01 M 2-inercapto- ethanol. Fractions, containing activity on depurinated DNA markedly higher than on intact DNA, were pooled and designated fractions IV A, B, C; these pooled fractions were concentrated in a hollow-fiber device to 10 ml and re-tested (Fig. 3). During the concentration procedure the activity of fraction TV A was lost. Fractions IVB and IVC remained active. The ratio of activity on depurinated DNA to intact D N A was established as 3.9 in the case of IVB and 55.0 for IVC.

The chemical homogeneity of fractions IVB and IVC was tested by gel chromatography and gel electrophoresis (Fig. 4). Both techniques showed that both samples are composed of two proteins although in different relative amounts. To determine the enzyme activity of both peaks, the acrylamide gel containing electrophoretically separated fraction TV C was sliced and the eluted proteins were tested on alkylated and depurinated D N A (Fig. 5). The large peak (cf. Fig. 4D, peak 1) revealed much higher specificity towards

i

6

5 c . _ E . 2 4 3 0

1

? I 3 pz 0

2

1

Fig. 3 . Eiizj'mir. ai.livity qf corzcenlnifed fi.aciion.v I V A , J V B arid I V C on iiitui.t and apurinic ("P]DNA.s. The while columns cor- respond lo intact, black ones to apurinic DNA. Activity was assaycd according to Paquette et al. [16]. The ratios of the activity on apurinic DNA to that on intact DNA were 3.9 for fraction 1VB and 55.0 for fraction IVC

depurinated DNA than the smaller peak (peak 2 in Fig. 4D).

The final step of enzyme purification was the chromatography of fraction IVC on a Sephadex G-150 fine column eluted with NaCl/Cit. The frac- tions of the large peak of protein (cf. fractions 21 -25 in Fig. 4C) were pooled, concentrated and analyzed for their homogeneity by gel electrophoresis in poly- acrylamide gel according to Davis [26]. Only one band was observed when 20 yg protein was applied on the gel and fractionated as described in legend to Fig. 4.

218 Barley Endonuclease Specific for DNA Apurinic Sites

0 I6 20 30 40 Tube number Migra t ion -

Fig. 4. G t ~ l c h r o m a t n ~ r u p h ~ ~ ( A , C ) artdgelelectrophoresi.s ( B , 0) offractions I V B ( A , B ) andIC’C: ( C , D j . (A, C) Gel chromatography: 0.5 ml of the sample was applied on a Sephadex G-150 fine column (1 x 50 cm), eluted with NaC1:Cit. Flow rate 1.8 ml . h-’, fraction volume 0.9 ml. (B, D) Gel electrophoresis: 400 PI (= 16 pg of proteins) of the sample was applied on the 7.5 polyacrylamide column (0.5 x 5 cm) and fractionated at 2 mA per column

9

8

7

6 - c - E

“ 5 1

c 3 0 0

1

1 4

0 m

3

2

1

C

Fig. 5. Gel rlt.ctr.opho~usis of frac~rion I V C : ussay ,fbr the enzymic activities. Conditions of gel electrophoresis as Fig. 4. Gels were sliced and slices eluted in 0.25 M Tris-HC1 buffer (pH 7.5), contain- ing 0.15 M KC1, for 36 h. The enzymic activity in the eluates was assayed according to Tomilin [4]. (------I A z ~ o ; (0-0) enzymic activity on alkylated [ th~mine-~HIDNA; (m-m) enzymic ac- tivity on apurinic [tl~ymine-~HH]DNA

This protein was used to determine the dependence of enzyme activity upon its concentration (Fig. 6A). At lower concentrations (up to 1.1 pg of protein in the reaction mixture) the enzyme is specific for de- purinated DNA under the conditions of the assay. It released a similar amount of radioactivity from depurinated DNA as was released from the same substrate by treatment with 0.3 M NaOH. In contrast, higher concentrations of the enzyme acted less specific- ally, releasing radioactivity from intact DNA and twice as much radioactivity from depurinated DNA as was released by NaOH (Fig. 6A). Incubation of intact DNA with NaOH (final concentration 0.2 M, 37 “C, 60 min) showed that this substrate contained no detectable apurinic sites. The pH optimum of the enzyme as determined on the depurinated substrate, is about 7.5 (Fig. 6B). The molecular weight estimated by gel chromatography and calculated from Ka, values is about 16000. According to the relative mobility of the single protein band obtained on dodecylsulphate/acrylamide gel electrophoresis, the molecular weight is estimated as 20000 (Table 1).

The yield of proteins in the purification procedure and degree of the purification are summarized in Table 2. Overall purification was 554-fold with a yield of 0.003 %.

J . Svachulovi, J. Satava, and J. Velcminsky 219

30 4

6.5 70 7.5 8 .O PH

Fig. 4. C'onceiitrciiiori depmrlmce rrnrlpH opiiiiniiii I:/ tlir endoinrc~lra,se specific /iir dty~urinufcd D N A . (A) Relation between the enzyme concentration and its activity on intact and depurinated DNAs. Fraction IVC was chromatographed on a Sephadex (3-150 fine column as described in Fig. 4, the protein peak was dialyzed against the assay buffer and concentrated. Different amounts of the concen- trated sample were assayed on intact (.----a) and depurinated (0 0) ["HIDNA 1161. Straight line (~----) represents the maximum releasc of acid-soluble radioactivity obtained by incuba- tion of depurinated substrate with 0.2 M NaOH (final concentra- tion) at 37 'C for 30 min. (€3) p H dependence ofthe enzymic activity. The aliquots of the fraction IVC, containing about 10 pg of the protein, were added to the reaction mixtures with depurinated DNA (total voluine 1 ml) of different pH and the enzymic activity followed according to Toinilin [4].

DISCUSSION

In this paper, the isolation of barley apurase is described. The process of its purification was partially based on the method used for the same enzyme isola- tion from calf thymus [19] and included streptomycin sulphate and ammonium sulphate precipitation fol- lowed by chromatography on phosphocellulose, hy- droxyapatite and Sephadex (3-150. Another pure plant apurase extracted from embryos of Pliaseolus nzultiflorus was purified using DEAE-cellulose and affinity chromatography [20]. The Phaseolus apurase is specific for DNA containing apurinic sites under all conditions tested. It possesses no exonuclease or glycosidase activity, is stimulated by Mg2+ and Mn2 ions, has a pH optimum in the range 7.5-8.0, and a molecular weight of40000. It is localized in chromatin

Table 1. Estimation of tnok~c~ul~ir weight hj' gel ./illration and ,yrl e l e c f ~ ~ ~ p l ~ o r e . ~ i . ~ A Sephadex (3-150 fine column (1 x 50 cm) was used with NaC1,'Cit as solvent. The reference samples (1.5 mg) were dissolved in 0.5 in1 NaCl/Cit. The same volume of concentrated enzyme was chromato- graphed in the same way. The elution was carried out at a flow rate of 3.4 ml . h-' and the proteins detected by their absorbance at 280 nm. The protein positions are expressed as K, , ( = V, ~ VO/ V , - V,, where V, is elution volume of a protein, VO is void volume and V, total volume of the column). Standards and samples for dodecylsulphate-gel electrophoresis were denatured by incubation with 1 y i sodium dodecylsulphate, 1 2-1nercaptoethanol, 8 M urea at 45 C lor 1 11. 100 pg protein was applied to the gel column and electrophoresis carried out at 6 mA per column. The positions of bands in gels are expressed in valucs of relative mobility ( R J as compared with broinphenol blue (R, = 1)

Sample Molecular K,, R , weight

Deoxyribonuclease I 31 000 - 0.545 25 000 0.48 - Ch ymotrypsinogen

Lysozyme 14 600 0.63 0.725 Cytochroine (' 12 270 0.71 -

Enzyme 0.61 0.645

Table 2. Purification of borlcy upurase A unit ol' apurase activity is defined as the amount of enzyme which causes 10 ".: release of acid-soluble radioactivity from the total substrate [20]. Specilic activity is expreased in unitdmg of protcin

Purification step Volume Total Total Specific protein activity activity

ml mg units units;mg

Crude extract 6150 7478 67 302 9 Ammonium sulphate

l'hosphocellulose

Fraction I V C

precipitation 235 761 27007 35.5

chromatography 215 108 8 100 75

froin hydroxy- apatite 44 0.88 1932 2195

Fraction 1VC after chromatography on Scphadcx (3-150 tine 2 0.22 1097 4990

whereas a different (not yet characterized) apurase is localized in the chloroplasts of Pliaseolus cells [20].

The barley apurase exhibits a similar specificity towards apurinic sites in concentrations up to 1.1 pg (5.5 units) per 10 pg depurinated DNA in the reaction mixture. At higher concentrations, i t degrades DNA non-specifically (Fig. 6A). Although polyacrylamide electrophoresis indicated the presence of only a single protein, we cannot exclude the possibility that the purified product contains a second; non-specific, nuclease in low concentration. Barley apurase is about half the molecular weight of those of E. coli

220 J. Svachulovi, J. Satava, and J. Veleminsky: Barley Endonuclease Specific for DNA Apurinic Sites

1171, Micrococcus luteus [13], calf thymus [19], human placenta [30] and Phaseolus nzultiflorus [20]. This striking difference was the main reason why we used two different methods to determine the molecular weight of two samples of independently isolated enzyme. In this respect, barley apurase is similar to several ultraviolet endonucleases like that isolated from T4 phage [31], from Micrococcus luteus [32] or from rat liver [33].

The major objective of this work was to isolate a pure enzyme. We did not characterize in detail other proteins obtained during the purification procedure. However, two other peaks from hydroxyapatite chro- matography (IVA, IVB), revealed higher affinity towards depurinated DNA (Fig. 2). The first one (IVA) was very unstable and was inactivated during the concentration process. Peak IVB was composed of two proteins with mobilities in polyacrylamide geIs slightly different to the proteins in peak IVC (Fig. 4). In another paper [21], we described two different fractions which were obtained from a DEAE- cellulose column during isolation of apurase from barley embryos. Both of these fractions were able to produce single-strand breaks in depurinated DNA but not in intact DNA. These facts lead us to suppose that, in common with E. coli [34], human placenta 1301 or calf thymus (T. Lindahl and J. Satava, un- published results), barley cells contain more than one apurase. The roles of these apurases in the repair of alkylated DNA in barley cells [23,24] remains to be determined.

We thank Prof. W. G. Verly (Liige). Prof. T. Lindahl and Dr P. Karran (Stockholm) for valuable comments on the manuscript.

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J . Svachulovi. J . Satava, and J . Veleminsky, Ustav Experimentilni Botaniky Ceskoslovenske Akademie VEd, Flemingovo nimcsti 2, CS-166 10 Praha, Czechoslovakia