Transcript
Page 1: Apurinic/apyrimidinic-specific endonuclease activities from Dictyostelium discoideum

304 Biochim.a et B.)phvs.'a A ~ta 824 ( 1 t;85) 304 312 Elsevier

BBA 91448

A p u r i n i c / a p y r i m i d i n i c - s p e c i f i c endonuc lease activit ies f rom

Dictyos te l ium discoideum

R o b e r t B. G u y e r and Reg ina ld A. Deer ing *

Molecular and Cell Biology Program, The Pennsylvania State UniversiO,. 201 Althouse Laboratot T, Universi(v Park'. PA 16802 (U,S.A.)

(Received December 10th, 1984)

Key words: AP endonuclease; DNA repair; (D. discoideum)

Two apurinic/apyrimidinic- (AP-) specific endonuclease activities have been isolated from the cells of Dictyostelium discoideum by fractionation on DEAE-cellulose, CM-cellulose and Sephadex G-75. These activities, designated A and B, have apparent molecular weights of 49 000 and 40 000, respectively. Although their precise reaction optima differ somewhat, both A and B quantitatively nick AP DNA best at pH 8.0-8.5 in low salt (less than 100 mM NaCI); both require Mg 2+. These activities are apparently specific only for AP sites in DNA. The low activities observed on heavily ultraviolet-irradiated DNA, gamma-irradiated DNA and osmium tetroxide-treated DNA are consistent with the small numbers of secondary AP sites expected in these DNAs. Both A and B produce single-strand nicks in AP DNA that result in termini that serve as good primers for Escherichia coli polymerase I. Hence, A and B appear to be Class II AP endonucleases which yield 3'-OH termini at nicks on the 5' side of baseless sugars. It is unclear whether A and B are independently coded proteins, different post-translational modifications of the same gene product, or whether one is an artifact arising from the isolation. Many of the properties of these D. discoideum AP endonuclease activities are similar to those of the predominant AP endonucleases observed in bacterial, plant and animal cells. They will be of use in the characterization of excision repair in this organism.

Introduction

DNA is altered in vivo by the inherent instabil- ity of certain bases, errors in replication and exog- enous damaging agents such as radiation and mutagenic chemicals. Excision repair is an im- portant mechanism for correcting these alter- ations. This is initiated by lesion-specific DNA glycosylases or endonucleases [1]. Several glyco- sylases have been described that remove specifi- cally damaged bases from DNA, producing an AP site [2]. This site can serve as the substrate for an

* To whom correspondence should be addressed. Abbreviations: AP, apurinic or apyrimidinic; Hepes, 4-(2-hy- droxyethyl)-i -piperazinethanesulfonic acid.

AP-specific endodeoxyribonuclease. Different AP endonucleases cleave in either of two ways, on the 3' side of the baseless sugar (Class I) or the 5' side (Class II) [3]. The 3'-OH terminus arising from the latter activity serves as a good primer for DNA polymerase I, whereas that from the former does not [4]. In some instances, the glycosylase and AP endonuclease activities are tightly associated, as for T4 endonuclease V and Micrococcus luteus ultraviolet damage-specific glycosylase/AP endo- nuclease [5-7]. Recently, Escherichia coli X-ray endonuclease (endonuclease III) and human lymphoblast endonuclease A have been shown to possess both glycosylase and AP endonuclease ac- tivities [8,9]. AP endonucleases with no associated glycosylase activity have been described for E. coli

0167-4781/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

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[10,11], M. luteus [12], Saccharomyces cerevisiae [13], Chlamydomonas [14], Phaseolus [15] and Drosophila [16]. In mammals, AP endonucleases from rat liver [17], calf thymus [18], human skin fibroblasts [19], placenta [20,21] and HeLa cells [22] have been described. Reviews of damage- specific glycosylases and endonucleases are availa- ble [2,3,11].

The cellular slime mold, Dictyostelium dis- coideum, has been used as a model system for the study of repair in simple eukaryotes [23,24]. Re- pair-deficient mutants sensitive to various damag- ing agents have been isolated [25-27]. Following ultraviolet irradiation, thymine dimers are re- moved from the DNA in vivo [24,28]. Here we report the isolation and characterization of two AP endonuclease activities from this organism.

Materials and Methods

Materials Chemicals werc from the following sources:

growth media (Difco); dithiothreitol, Hepes, poly(ethylene glycol) (Mr approx. 8000), sodium lauroyl sarcosine, bovine serum albumin, pepsta- tin, leupeptin, ovalbumin, soybean trypsin inhibi- tor, dATP, dCTP and dGTP (Sigma); Dextran T-500 and Sephadex G-75 (10-40 /xm) (Pharmacia); DEAE-cellulose 32 and CM-cellulose 52 (Whatman); agarose (Standard, low Mr) and Protein Assay Dye Reagent (Bio-Rad); ethidium bromide (Calbiochem-Behring); osmium tetroxide, crystalline (Polysciences); bovine pancreatic DNAase I, crystalline, 2064 U/mg (Worthington); E. coli DNA polymerase I, endonuclease-free (Boehringer Mannheim); and [y-32p]dTTP (600 Ci/mmol) (New England Nuclear).

All buffers for columns and reactions were pre- pared in autoclaved containers using autoclaved, double-distilled H20 to minimize contamination by other nucleases. The pH values of all solutions were determined at room temperature. All steps in the enzyme purifications were performed at 0-4°C. The proteinase inhibitors pepstatin (1/~g/ml) and leupeptin (5 #g/ml) were present at cell lysis and all stages of purification, storage and assay. These inhibitors and the maintenance of a nonacid pH may have provided some protection against the action of the proteinases of D. discoideum [29,30].

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These inhibitors had no adverse effects on the endonuclease activities under investigation.

Preparation of the extract An axenic, repair-proficient strain of D. dis-

coideum, NP-2, was germinated from spores [23,25] and maintained in HL-5 medium [31]. For enzyme isolations, 4 1 of cells were grown for about 60 h in a 7.5-1 New Brunswick Fermentor at 23°C and harvested at mid-to-late log phase ((4-8).106 cells/ml) by centrifugation at 400 x g for 5 min. The 20-24 g cell pellet was washed three times by suspension in 250 ml phosphate-buffered saline (34 mM KH2PO4/16 mM Na2HPO4/9.9 mM NaC1/10 mM KC1, pH 6.5) [25] and centrifuga- tion. The washed cells were suspended at about 2.5.108 cells/ml in ice-cold, 50 mM Tris/5000 mM NaC1 (pH 8.0) [7], and sonicated for 5 x 60 s at 0°C, using a Fisher Model 300 Sonic Dismem- brator with a 19-mm diameter tip. The super- natant (about 70 ml) from a 90-min centrifugation at 65 000 x g was adjusted to 6% (w/v) poly(ethyl- ene glycol) and 4% (w/v) Dextran T-500 in 40 mM Tris/4000 mM NaC1 (pH 8.0), followed by gentle stirring for 2 h at 4°C and centrifugation at 20 000 x g for 20 min [7]. The top (poly(ethylene glycol)) phase was saved; the lower (dextran) r" ase was reextracted with the top phase from a 'wash' mixture (35 ml of 40 mM Tris/4000 mM NaC1, pH 8.0), adjusted to 6% (w/v) poly(ethylene gly- col) and 4% (w/v) Dextran T-500, stirred 2 h and centrifuged. Following stirring for 1 h and centri- fugation, this second poly(ethylene glycol) phase was pooled with the first poly(ethylene glycol) extract phase [7,32]. These combined poly(ethylene glycol) phases were dialyzed overnight against two changes of 6 1 of 10 mM Tris-HC1 (pH 8.0)/0.1 mM dithiothreitol/1.0 mM EDTA/10% (v/v) eth- ylene glycol (buffer A) containing 5% (w/v) poly(ethylene glycol). A small amount of precipi- tate was removed by centrifugation and discarded.

D EAE-cellulose chromatography A column (2.5 x 33 cm) of DE-32 was equi-

librated in buffer A [7,18,32], and the dialyzed and centrifuged poly(ethylene glycol) extract super- natant was applied at 60 ml/h. The column was washed until the refractive index (due to poly(eth- ylene glycol)) returned to baseline and was devel-

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oped with a 1200-ml linear gradient from 0-500 mM NaCl in buffer A. Column eluant was passed through an ISCO ultraviolet monitor (280 nm, 5 mm pathlength) and dripped into fraction collec- tor tubes containing pepstatin and leupeptin. The final concentrations of pepstatin and leupeptin in the 10-ml fractions were 1 and 5 ~g/ml, respec- tively. Pooled fractions were concentrated about 6-fold by ultrafiltration in a 50-ml, stirred cell containing a PM-10 membrane (Amicon).

CM-cellulose chromatography A column (1.5 × 23 cm) of CM-52 was equi-

librated in 10 mM NaH2PO4/10 mM CHBCOONa/0.1 mM dithiothreitol/1.0 mM EDTA/10% (v/v) ethylene glycol (pH 5.50) (buffer B). The 9-ml sample, adjusted to pH 5.5, dialyzed 2-4 h against buffer B, followed by centrifugation, was applied at 30 ml/h, and the column was developed with a 350-ml linear gradient, to 600 mM NaC1 in buffer B. Eluant was dripped into tubes containing pepstatin and leupeptin and enough 200 mM NazHPO 4 (adjusted to pH 8.0 with HC1) buffer to maintain sample pH at >/7.0. Pooled fractions were concentrated about 10-fold to 2 ml.

Sephadex G-75 A column (1.6 × 93 cm) of Sephadex G-75 was

equilibrated with 10 mM Hepes/500 mM NaC1/0.1 mM dithiothreitol/1.0 mM EDTA/10% (v/v) ethylene glycol (pH 8.0) (buffer C). A 1-ml sample from the CM-cellulose pooled concentrate was applied and eluted at 3.45 ml/h. The column was calibrated with bovine serum albumin, ovalbumin and soybean trypsin inhibitor.

Preparation of native and damaged PM2 DNA Covalently closed circular duplex PM2 DNA

was purified from a 20-1 culture as described by Espejo and Canelo [34] with recent modifications [35]. This PM2 DNA (approx. 600 /xg/ml) was stored in 10 mM Tris/200 mM NaC1/2 mM EDTA (pH 8.3) at -75°C.

AP DNA was prepared by heating PM2 DNA in 10 mM sodium citrate/100 mM NaC1 (pH 5.0) at 70°C for 5 min/AP site per DNA molecule [36]. The AP DNA was kept refrigerated at pH approx. 7.5 in the presence of 1 mM EDTA.

Ultraviolet-irradiated PM2 DNA was prepared at room temperature by irradiating 50-t~l aliquots with 254 nm light at a dose rate of 3 5 J /m 2 per s (Model J225 dosimeter, UltraViolet Products) [35]. The ultraviolet-irradiated DNA (150 t~g/ml) in 3 mM Tris/55 mM NaCI/1 mM EDTA (pH ap- prox. 7.5) was stored at 4°C. The number of pyrimidine dimers in ultraviolet-irradiated PM2 DNA was calculated from the published data of Woodworth-Gutai et al. [37].

Gamma-irradiated PM2 DNA was prepared by irradiation of DNA with 6°Co gamma rays at 0°C in 10 mM Tris/1 mM EDTA/50 mM KI (pH 8.0) [38]. The dose rate in the Gammacell 200 (Atomic Energy of Canada) was 8.2 krad/min. The 50 t~g/ml solution of gamma-irradiated DNA was dialyzed against 10 mM Hepes/50 mM NaCI/5 mM MgC12 (pH 8.0) (buffer D).

Osmium tetroxide-treated PM2 DNA was pre- pared by the method of Armel et al. [38]. Briefly, PM2 DNA (50 ~g/ml) was dialyzed extensively against 0.12 mM sodium cacodylate (pH 7.0). The DNA was heated to 65°C for 10 rain as was an equal volume of freshly diluted 0.2% osmium tetroxide in the same buffer. Then the osmium tetroxide was added to the DNA and the mixture was incubated for 15 rain at 65°C. Four extrac- tions (of osmium tetroxide) with ice-cold diethyl ether were followed by exhaustive dialysis of the aqueous phase against 10 mM Tris/1 mM EDTA (pH 8.0)[38].

Endonuclease nicking assay Unless otherwise noted, the conditions listed in

this section apply to all assays (i.e., standard as- say) in this report. Buffer D (see above) was the usual assay buffer. The DNA storage solution contributed 7 mM NaC1 to the reactions. PM2 DNA and damaged DNAs were diluted to 20 /~g/ml in buffer D containing 100/~g bovine serum albumin/ml. The bovine serum albumin was pre- viously heat-treated at 100°C for 5 rain at pH 3.0 to minimize the activity of any contaminating nucleases [39]. The substrate for the standard AP endonuclease assays was PM2 DNA containing six AP sites/molecule. Enzyme samples were diluted in buffer D containing 1 /~g pepstatin/ml, 5 /~g leupeptin/ml, 500 t~g heat-treated bovine serum albumin/ml and 10% (v/v) ethylene glycol (buffer

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E). The reaction mixture contained 30 #1 buffer D, 20/tl (0.40 #g) DNA and 20 ~tl enzyme. After 60 rain at 30°C, the reaction was stopped by adding 20 Itl of 10% (w/v) sodium lauroyl sarcosine/30% (w/v) sucrose/0.03% bromophenol blue.

A 45-/~1 aliquot of the stopped reaction mixture was applied to wells of a horizontal 1% agarose gel (20 × 25 cm; Bethesda Research Laboratories). The unnicked DNA (Form I) was separated from the nicked species (Form II, open circular or Form III, linear) by electrophoresis at 50 V for 12-18 h [40,41]. The electrophoresis buffer (50 mM Tris/20 mM sodium acetate/18 mM NaCI/2 mM EDTA (adjusted to pH 8.0 with acetic acid) [42] was recycled between buffer chambers at approx. 1 1/h. Gels were stained for 45 min in the dark in 1100 ml of electrophoresis buffer containing 1 /~g ethidium bromide/ml, illuminated with 302 nm light (Fotodyne), and photographed through glass and Wratten 2B and 29 filters [41,43]. The Kodak Tri-X film was developed according to the manu- facturer's instructions, and the density of entire bands was measured with a Quick Scan Jr TLC unit (Helena). The densities were linear for 0-0.20 #g DNA/band. The nicking activity (average number of single-strand breaks/DNA molecule) was calculated, assuming the Poisson distribution of nicks, as - I n of the proportion of superhelical molecules remaining unnicked [40]. The product of the nicking of Form I DNA was predominantly Form II. However, a small amount of Form III DNA was observed with reactions that produced more than approx. 1.5 nicks/DNA molecule. One unit of activity was defined as the amount of enzyme that hydrolyzed one femtomole of phos- phodiester bonds per minute under the standard assay conditions described above.

Protein assay The protein concentration of enzyme samples

was determined by the method of Bradford [44] using ovalbumin as the standard.

DNA polymerase assay The determination of whether nicks were pro-

duced on the 3' or 5' side of the AP site was performed as described by Mosbaugh and Linn [4]. The PM2 DNA had an average of 1.5 AP sites/molecule. Bovine pancreatic DNAase I was used as a positive control [41].

Results

Purification of the enzyme(s) The elution profile of a typical DEAE-cellulose

column is shown in Fig. 1. Two peaks of AP endonuclease activity, designated 'A (fractions 20-25) and B (fractions 31-36), eluted early in the gradient along with considerable amounts of other proteins. Since these fractions were assayed in 57 mM NaC1, activity B may have been under- estimated by about 50% (see Fig. 5). Also, two peaks of activity that nicked undamaged PM2 DNA at pH 7-9 eluted in fractions 13-19 and 24-33 (not shown). Most of these nonspecific ac- tivities eluted before peak A and between peaks A and B. In addition, a major nonspecific endo- nuclease activity that was active at acid pH eluted in fractions 56-64 (Guyer, R.B., Skantar, A.M. and Deering, R.A., unpublished data).

Peaks A and B from the DEAE-cellulose col- umn were concentrated and fractionated on CM- cellulose. Fig. 2 shows a composite of column runs for peaks A and B. The protein content of peaks A (fractions 45-50) and B (fractions 42-47) decreased dramatically during the purification on CM-cellulose (Table I). The contaminating non- specific endonuclease activities also decreased.

Peaks A and B from the CM-cellulose columns

l i t I A I B i I I ,

! `.°

-->" )'1 t " 1 ~°° ] ,; I L _ . - - " / T ~ , - , ~ ,~ 6 I I. . . . - " - J I

"~ I', ,1~ J .4 ' , ....-',.. 7 ~"

-~ / "1 ~ , A " K_ . . . . , - ~ i ~

oLj-j "1o 30 50 70 Fraction Number

"7 1.0 ,

0

3.5 *~ c

.D

Fig. 1. DEAE-cellulose chromatgraphy of the poly(ethylene glycol) phase of a crude extract from D. discoideum cells. The eluant flowed through an ultraviolet monitor (ISCO UA-5, 5 m m pathlength) to measure the absorbance at 280 nm ( - - - - - ) . The concentration of NaCI ( . . . . . ) in the 10-ml fractions was determined by refractive index. The AP endo- nuclease activity was determined as described in Materials and Methods and plotted as relative activity (© O). The highest activity (relative activity = 1.0) was 1.67 units in 20 ~1 of a 1:18000 dilution of fraction 32.

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4 / I I I [ 1 I

I.Q- ~2

300 i

. 1 - - ~

0.5 ~ - ~ ~ :,

Z

I0O

0 40 50 60

Fraction Number

Fig. 2. CM-cellulose chromatography of activity peaks A and B from the DEAE-cellulose column. The concentration of NaCI ( . . . . . ) in the 3.5-ml fractions was determined by refrac- tive index. This figure is a composite of separate runs for peaks A and B, and the peaks are labeled as in Fig. 1. The AP endonuclease activity was determined for peak A (e e) and B (O ........ O) as described in Materials and Methods. The highest activity (relative activity = 1.0) for peak B was fraction 44 (1.87 units in 20 #1 of a 1 : 7500 dilution) and for Peak A was fraction 48 (2.01 units in 20 #1 of a 1:10000 dilution).

were concentrated and further purified on Sep- hadex G-75. The protein content for both A and B decreased and the specific activity (units/rag

protein) increased. After Sephadex G-75 purifica- tion, the nonspecific endonuclease activity was not measurable in A or B at the dilutions used for assaying the AP endonuclease activities. The aver- age of two determinations gave molecular weights of 49000 and 40000 for A and B, respectively. The purification of AP endonuclease activities A and B is summarized in Table 1.

Temperature optimum The reaction rates of AP endonuclease activities

A and B exhibited identical temperature profiles with the peak at 30°C. The reaction rates de- creased to approx. 50% at 15 and 37°C.

pH optimum The pH profiles for activities A and B are

shown in Fig. 3. Both activities were highest be- tween pH 7.5 and pH 8.5, with a peak at about pH 8.0.

Divalent cations

A P e n d o n u c l e a s e a c t i v i t i e s A a n d B h a d a

n e a r l y a b s o l u t e r e q u i r e m e n t f o r M g 2 + ; a p p r o x .

1 5 0 0 - t i m e s m o r e e n z y m e w a s n e e d e d in 10 m M

E D T A to p r o d u c e t h e s a m e n u m b e r o f n i c k s in A P

TABLE I

PURIFICATION OF AP ENDONUCLEASE ACTIVITIES A AND B

The results of a typical purification from 4 1 of D. discoideum NP-2 cells are presented. One unit of AP endonuclease activity is defined as the amount of enzyme that hydrolyzed one femtomole of phosphodiester bonds per minute.

Fraction Total AP-endo- Total Specific activity Purification nuclease protein (uni ts /mg protein) factor b activity (mg) ( × 10- 3) (-fold) (units)( x 10 - 6 )

I. Extract super- natant a approx. 95 3800 approx. 25 1.0

II. Poly(ethylene glycol) phase a approx. 125 2100 approx. 60 approx. 2.4

A B A B A B A B

III. DEAE-cellulose 31 54 170 160 180 340 7.2 14 IV. CM-cellulose 16 11 0.88 0.31 18000 35 000 720 1400 V. Sephadex G-75 11 4.7 0.06 ~ 0.01 c 180000 470000 7200 19000

a There is some uncertainty in the enzyme activities in the extract supernatant and the poly(ethylene glycol) phase due to the contaminating 10-20% nonspecific endonuclease activity and to the instability of the AP endonuclease(s) in these crude preparations.

b The purification factors may be somewhat high, since they are based on low (unstable) enzyme activity in the extract supernatant. c Protein content was at the lower limit for detection in the assay.

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~ 1.0

I

T_- 0.5

I I I 1 I

I I I I I 7.0 8.0 9,0

pH

Fig. 3. Dependence of reaction rate (relative activity) on pH for AP endonuclease activities A and B after their final purifica- tion on Sepbadex G-75. The substrate was AP PM2 D N A (six AP sites/molecule). The reactions were for 60 rain at 30°C (see Materials and Methods). The buffer for pH values 7.0-8.5 was 10 mM Hepes, 50 mM NaCI, 5 mM Mg 2÷, while 10 mM Tris was substituted for Hepes for a duplicate of pH 8.5, and for pH 9.0 and 9.5. The highest reaction rate (average number of n i c k s / D N A molecule per h) for each enzyme activity (A = 1.36, B = 1.35) was used as relative activity = 1.0).

DNA as in 1-5 mM Mg 2+. The data in Fig. 4 show the relative reaction rates of activities A and B at various concentrations of Mg 2+ in 10 mM Hepes/57 mM NaCI (pH 8.0). Activity A was highest between 5 and 10 mM Mg 2÷, while activ- ity B had a peak at 1 mM Mg 2÷ followed by a decline at higher Mg 2÷ concentrations. When 5 mM Mn 2÷ was substituted for 5 mM Mg 2÷, the

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reaction rates for both A and B were only 30% of those in Mg 2+. Substitution of 5 mM Ca 2+ for 5 mM Mg 2÷ resulted in only 10% of the activity seen in 5 mM Mg 2÷.

Monooalent cations The effect of NaCI concentration on the reac-

tion rate is shown in Fig. 5. Before the addition of more NaC1, the basic reaction mixture contained 10 mM Hepes/7 mM NaC1/5 mM MgC12 (pH 8.0). Activity A was highest at NaC1 concentra- tions of less than 107 mM, with a peak at about 57 mM NaCI. Activity B exhibited maximal activity at 7 mM NaCI and declined to 50% at 57 mM NaC1. KC1 could be substituted for NaC1 at the concentrations shown in Fig. 5.

Linearity of initial reaction rates Except for those leading to the data shown in

Table II, all reaction mixtures reported here con- tained a limiting amount of enzyme and 0.40 #g DNA with an average of six AP sites/molecule. The reactions were linear in the range of 0.2-2.3 average nicks/DNA molecule. The amount of en- zyme was varied to keep the number of nicks in the linear range. Reactions were linear with time and with the number of AP sites/molecule used in the standard assay.

I I ~ a I i a 11 i i

0 5 I0 20 [MgZ+](mM)

Fig. 4. Dependence of reaction rate on Mg 2+ for purified AP endonuclease activities A and B. The buffer was 10 mM Hepes, 50 m M NaCl (pH 8.0), with the indicated concentra- tions of Mg 2+. More details are given in Materials and Meth- ods. The highest reaction rate (A = 0.95, B = 2.10 average number of n i c k s / D N A molecule per h) for each AP endo- nuclease activity was used as relative activity = 1.0.

1.0

0.5

o g~

I I I I I I

0 ~ I I I I I I 50 I00 t 50

[NoCI] (raM)

Fig. 5. Dependence of reaction rate on concentration of NaCI for purified AP endonuclease activities A and B. The buffer was 10 mM Hopes, 5 m M Mg 2+ (pH 8.0), with the indicated concentrations of NaC1. More details are given in Materials and Methods. The highest reaction rate for each AP endo- nuclease activity ( A = 0 . 9 8 , B = 2.28 average number of n i c k s / D N A molecule per h) was used as relative activity = 1.0.

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TABLE II

ACTIVITY OF AP ENDONUCLEASE A AND B UPON DAMAGED DNAS

The preparation of substrates and reaction conditions are described in Materials and Methods. All reactions used for this table were for 60 min and contained excess enzyme so that reactions with AP DNA were complete within 30 rain. Any nonspecific nicking has been subtracted in the calculations for the damaged PM2 DNAs.

Substrate Average number of nicks formed/DNA molecule

A B

Untreated DNA (native) AP DNA (1.50 AP sites/molecule) Ultraviolet-irradiated DNA (50 j/m2;

10 pyrimidine dimers/molecule) Ultraviolet-irradiated DNA (1200 J/m2;

230 pyrimidine dimers/molecule) Osmium tetroxide-treated DNA (0.1% OsO4,

65°C, 15 min; > 3 damage sites/molecule) Gamma-irradiated DNA (16 krad, in 50 mM

KI; about 0.4 AP sites, plus other damage)

< 0.05 a < 0.05 1.41 1.41

0.05 0.05

0.21 0.14

0.44 0.44

0.39 0.28

a The lower limit of detection was 0.05 nicks/DNA molecule, and the lower limit of reproducible results was about 0.2 nicks/DNA molecule.

Stabifity of AP endonuclease activities A and B The enzyme activities were stable at 4°C after

fractionation on the DEAE-cellulose column and in the later stages of purification. Storage solutions were 10 mM Hepes or Tris-HC1 buffer (pH 7-8) and contained 10% (v/v) ethylene glycol, 1 /~g pepstatin/ml, 5 /~g leupeptin/ml and the NaC1 (and other additives) used for the column elutions. Eluted fractions from the CM-cellulose and the Sephadex G-75 retained more than 80% of their activities after 3 months of storage at 4 or - 20°C. The addition of ethylene glycol to 35% (v/v) re- sulted in the same stability at 4 and - 2 0 ° C .

Activity of AP endonuclease(s) upon damaged DNAs Even with enzyme excess, activity against un-

damaged DNA was barely detectable (Table II). With DNA containing 1.50 AP sites/molecule, both activities A and B produced an average of 1.41 n i cks /DNA molecule, indicating essentially one nick per AP site. When DNA was treated with 254-nm light to p roduce 10 pyr imidine d imer s /DNA molecule, only barely measurable nicking occurred, indicating that activities A and B did not recognize pyrimidine dimers. With heavily ultraviolet-irradiated DNA (1200 j /m2) , there was a measurable increase in nicking activ-

ity, but nicks were only 0.06-0.09% of the number of pyrimidine dimers.

Both activities A and B produced a maximum of 0.44 n i c k s / D N A molecule in osmium tetroxide-treated DNA (Table II) which contained considerably more than three damaged sites per molecule [38]. AP endonuclease activities A and B produced 0.39 and 0.28 n i cks /DNA molecule, re- spectively, in DNA that was exposed to a dose of 16 krad of 6°Co gamma rays (Table II).

Nature of the incision site in AP DNA When PM2 DNA with 1.5 AP sites/genome

was nicked with AP endonuclease activities A and B, the product supported DNA synthesis by E. coli DNA polymerase I. The rates of synthesis in DNA nicked by A and B were nearly identical to that in DNA nicked to the same degree by pan- creatic DNAase I, which yields a good primer [41]. The slopes of the curves for the incorporation of [32p]dTTP into the DNA vs. time were similar to previously published values [4,9].

D i s c u s s i o n

We have purified specific and stable AP endo- nuclease activities from D. discoideum. The insta-

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bility observed in the initial lysates and in the early stages of purification are probably due to proteolytic degradation by the highly active pro- teinases of this organism [30]. Two peaks of activ- ity (A and B) were resolved and purified on DEAE-cellulose, CM-cellulose, and Sephadex G-75 columns. Although these activities display some- what different apparent molecular weights (A, 49000; B, 40000) and profiles for optimum reac- tion conditions (Fig. 3, pH; Fig. 4, Mg2+; Fig. 5, NaC1), it is unclear whether they are indepen- dently coded proteins, post-translationally mod- ified, or proteolytically degraded forms of the same gene product. Studies of AP-negative mutants and/or antigenic properties would help clarify this uncertainty. The major AP activities of human placenta and rat liver were found to display several chromatographic forms [20,45]. Recently the major portion of these mammalian activities was shown to reside in one homogeneous protein in each cell type; the previously observed multiple forms prob- ably resulted from proteolytic degradation of one major AP endonuclease [17,21].

Our results are consistent with the conclusion that both the A and B activities are specific for AP sites in DNA. The AP sites in acid/heat-treated PM2 DNA were quantitatively nicked when the reaction was run to completion (Table II). In addition, at low enzyme concentrations, the initial rate of nicking was a linear function of the number of AP sites/DNA molecule. The agarose gel elec- trophoresis patterns after the reactions indicated that most of the nicked DNA was Form II (open circular), showing the production of predomi- nantly single-strand nicks. At the enzyme con- centrations used for these quantitative studies, lit- tle if any nonspecific endonuclease activity was observed. PM2 DNA that was irradiated with ultraviolet light served only very weakly as a sub- strate, with the limiting number of single-strand scissions amounting to less than 1% of the number of cyclobutane pyrimidine dimers. The slight activ- ity on ultraviolet-irradiated DNA may have been due to a small number of AP sites resulting from loss of altered (non-dimer) bases [13,46]. Although the situation with osmium tetroxide-treated or gamma-irradiated DNA was not quite as definitive as for ultraviolet-irradiated DNA, the results were nevertheless consistent only with AP sites serving

311

as substrate in these cases also. The observed maximum numbers of nicks in these latter DNAs (Table II) is certainly far less than would be expected if thymine glycols or other base alter- ations were acting as sites for scission by our purified activities; it is instead in the order of the number of AP sites expected in these damaged DNAs [38,47,48]. Therefore we conclude that we have identified an endonuclease(s) specific for AP sites, with little, if any, associated glycosylase or base damage-specific nicking activity.

The nicked AP DNAs produced by our two endonuclease activities (A and B) served as sub- strate for nick translation by DNA polymerase I to about the same degree as DNA containing the same number of nicks produced by pancreatic DNAase I. We conclude that the observed AP- specific nicking was occurring on the 5' side of the baseless sugar, yielding a 3'-OH terminus that was a suitable primer for polymerase I [4]. Hence the A and B activities appear to be Class II AP endo- nucleases [3]. These yield termini directly useable in subsequent repair steps of 5' to 3' strand elon- gation and 5' to 3' exonucleolytic excision of damage ('repair synthesis', 'nick translation') without the requirement for the removal of a base- less sugar at the 3' end before polymerization can start. The dominant AP activities in most cells appear to be Class II [2,3,11]. Class I activities are often found associated with glycosylases [3].

The AP activities from D. discoideum bear con- siderable similarities to those from other organisms. The molecular weights of 40000 and 49000 are similar to those reported for several organisms which are all in the molecular weight range of 30000-42000. Many (but not all) require Mg 2+, as is the case with D. discoideum activities A and B (Fig. 4). The pH optima of most reported AP endonucleases are in the range of 7-9, generally about 8.0-8.5; this is also the optimum for the D. discoideum activities (Fig. 3). Many are inhibited by high (0.4-0.5 M) NaCI concentrations, as are those of D. discoideum (Fig. 5). (For a com- pendium of sources and properties of various AP endonucleases, see Friedberg et al. [11].

These highly purified (up to almost 19 000-fold; Table I) AP-specific activities can serve as useful reagents in the search for other repair enzymes (such as glycosylases). They can also provide the

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312

basis for comparing AP activities in repair mutants with those in the repair-proficient, wild-type cells of D. discoideum.

Acknowledgments

This investigation was supported by U.S. Public Health Services research grant GMl6620 from the National Institute of General Medical Sciences. We thank Kathleen Ankers, James Nonnemaker and Andrea Skantar for excellent technical assis- tance.

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