Mutation Research, DNA Repair, 254 (1991) 263-272 © 1991 Elsevier Science Publishers B.V. 0921-8777/91/$03.50 ADONIS 0921877791000708

263

MTDNA 06433

AP endonuclease and uracil DNA glycosylase activities in Deinococcus radiodurans C. Ian Masters a, Bevan E.B. Moseley b and Kenneth W. Minton

a

a Department of Pathology, F.E. H~bert School of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799 (U.S.A.) and b Institute of Food Research, Reading Laboratory, Shinfield, Reading, Berks., RG2 9A T (Great Britain) (Received 13 September 1990) (Revision received 3 December 1990) (Accepted 4 December 1990)

Keywords: Deinococcus radiodurans; AP endonuclease; Uracil DNA glycosylase; Base excision repair

Summary An endonuclease specific for apurinic/apyrimidinic (AP) sites was identified and purified from extracts of Deinococcus radiodurans. The enzyme is 34.5 kD, has no activity towards normal, alkylated, uracil-containing, or UV-irradiated DNA, and is active in the presence of EDTA. The addition of up to 10 mM Mg 2+ or Mn 2+ did not affect activity, but higher concentrations were inhibitory. There is no associated exonuclease activity, either in the presence or absence of divalent cation. Optimal reaction conditions were 150 mM NaC1 and pH 7.5. A uracil DNA glycosylase was also detected, active in the presence of EDTA, selectively removing uracil from DNA without generating other byproducts. The optimal reaction conditions were 50 mM NaC1 and pH 7.5. Implications for base excision repair in D. radiodurans are discussed.

The genus Deinococcus is composed of four species that are extremely resistant to the lethal and mutagenic effects of many agents that damage DNA (for review see Moseley, 1983). The most studied of these species is D. radiodurans, in part because it is naturally transformable (Tirgari and Moseley, 1980), a property that has facilitated efforts at genetic characterization (Moseley, 1983; Smith et al., 1988, 1989; Lennon and Minton,

Correspondence: Dr. Kenneth W. Minton, Department of Pathology, F.E. H6bert School of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799 (U.S.A.), Tel. (301) 295-3476.

1990). Although repair of DNA damage by D. radiodurans is known to be unusually efficient, the

molecular mechanisms responsible remain largely unidentified (Moseley, 1983). On the basis of in vivo and in vitro evidence only two types of repair have been detected in D. radiodurans: nucleotide excision repair and recombinational repair (Moseley, 1983). Only two DNA-repair enzymes have thus far been documented, UV endonuclease-a, on the basis of in vivo evidence, and UV endonuclease-fl, which has been detected both in vivo and in vitro (Evans and Moseley, 1983, 1985; Moseley and Evans, 1983). Both enzymes incise UV-irradiated DNA and both are thought to be endonucleases, as opposed to glycosylases: in the

264 case of UV endonuclease-a because of its broad substrate range; and in the case of UV endonuclease-fl because glycosylase activity is absent in preparations of purified enzyme (Evans and Moseley, 1988). Described here are two additional DNA-repair activities detected in crude extracts of D. radiodurans and partially purified: an AP endonuclease and uracil DNA glycosylase. In combination, their presence suggests the occurrence of base-excision repair, a process not previously demonstrated in this remarkably DNA-damage resistant organism. Materials and methods

Bacterial strains, growth and transformation Deinococcus radiodurans strain R1 (wild-type; Brooks and Murray, 1981) and strain 78 (mtcA- uosE- derivative of strain R1; Moseley and Evans, 1983) were grown in TGY nutrient broth (0.8% tryptone, 0.1% glucose; 0.4% yeast extract) or TGY plates, solidified with 1.5% agar. Escherichia coli was grown at 37 ° C in LB broth or on LB plates containing 1.5% agar. Transformation procedures for D. radiodurans were described previously (Smith et al., 1988). Briefly, 0.1 ml of competent D. radiodurans recipients was transformed with 1 /~g DNA and then diluted 10-fold and incubated at 32°C overnight to permit expression prior to plating on selective agar containing 0.03 /~g/ml mitomycin C (MMC). Transformation of E. coli was by the CaC12 method. To incorporate uracil into plasmid DNA the plasmid was grown in E. coli CJ236 (dut ung; Bio-Rad). Otherwise plasmids were propagated in E. coli DH5a (BRL). Preparation of radiolabelled DNA (1) E. coli B / C 2 / H T (thy ; Evans, 1984) was ~Hrown in M9 medium containing 1 /xCi/ml [Me]thymidine and chromosomal DNA purified as described (Marmur, 1961). The specific activity of the DNA was 1 × 10 4 cpm//~g. (2) Salmon sperm DNA was labeled with [Me3H]thymine or [5-3H]uracil by nick-translation using [Me-3H]'I-TP or d[5-3H]UTP in place of TFP, respectively. The specific activity in both cases was 2 × 103 cpm//~g.

Alkylation and depurination of radiolabelled E. coli chromosomal DNA E. co# chromosomal DNA containing [Me3H]thymine was alkylated and alkylated-depurinated according to Verly (1981). Alkylation employed methyl methanesulfonate (MMS) yielding ca. 1 methylation per 6-7 nucleotides (Verly, 1981). To generate highly depurinated DNA, the E. coli chromosomal DNA was first alkylated, as above, and then depurinated by incubation at 5 0 ° C for 6 h followed by dialysis against SSC (150 mM NaCl, 15 mM Na citrate, pH 7.0) at 4 ° C. This treatment generates ca. 1 AP site per 20 nucleotides (Verly, 1981). Depurination, alkylation and deamination of pAT153 pAT153 is a 3.7 kb, Ap R, Tc R, deletion derivative of pBR322 (Twigg and Sherratt, 1980). pAT153 (neither radiolabelled nor alkylated) was depurinated by the procedure of Teebor and Brent (1981) by heating at pH 4.4. The method produces about one depurination per 3000 nucleotides. For alkylation of pAT153, 50 /~g of plasmid was incubated with 12 mM MMS and 10 mM Tris-HC1, pH 7.5, in a total volume of 110 /~1 at 37 ° C for 20 min. The DNA was then ethanol-precipitated twice and resuspended in 10 mM TrisHC1, pH 7.5. Deamination of cytosines to form uracil in pAT153 was performed by treatment with bisulphite under anaerobic conditions according to Lindahl et al. (1977), using conditions that produce 1-2 uracil residues per plasmid. Assays for AP endonuclease (1) Conversion of supercoiled to open circular pAT153 was assayed by agarose gel electrophoresis. Standard reaction conditions were 1 ~tg pAT153 incubated with enzyme fraction, 10 mM Tris-HC1, pI-I 7.0, 50 m M NaC1 and 1 mM EDTA in a total volume of 20/~1 for 30 min at 37 ° C, and terminated by the addition of 1 /xl containing 20 /Lg sodium dodecyl sulfate (SDS), 10 /~g EDTA, 200 /~g ficoll, 10 #g bromophenol blue, and 5 /~g tris base, followed immediately by agarose gel electrophoresis. One unit of AP endonuclease activity was defined as the activity required to con-

265 vert 1 /~g of depurinated supercoiled plasmid to open circular form under the reaction conditions above. (2) Release of acid-soluble label employed as substrate E. coil chromosomal DNA containing [Me-3H]thymine (1 × 104 cpm//~g). In a typical reaction, 1 /~g of radiolabelled E. coli DNA was incubated with enzyme fraction in SSC, pH 7.0, 0.2 mM EDTA in a total volume of 40/~1 at 37 ° C for 30 min. The reaction was then quenched in ice, 100/~1 of 2 mg/ml calf-thymus DNA in 0.1 × SSC and 500 btl of 6.4% perchloric acid were added, mixed and left in ice for 15 min, prior to centrifugation. Radioactivity in an aliquot of supernatant was determined by scintillation counting. In both assays E. coli exonuclease III, incubated in the presence of 5 mM Ca 2+, was employed as a positive control. In the presence of Ca 2÷ exonuclease III shows only AP endonuclease activity (Rogers and Weiss, 1980).

Assay for uracil DNA glycosylase Standard reaction conditions were as follows: A total reaction volume of 40/xl contained 10 mM Tris-HC1, pH 7.6, 2 mM EDTA, 50 mM NaC1, 0.3 /~g of [5-3H]uracil-containing salmon sperm DNA (6 × 104 cpm) and 2 /~1 (16000 units) of uracil DNA glycosylase-containing cell free extract, incubated at 37°C for 15 min. For chromatography, reactions were quenched in ice and 2 /zl of the reaction mixture immediately spotted and dried on the appropriate paper or thin-layer plate. For measurement of perchloric acid-soluble label, the reaction was terminated and radioactivity assessed as described above for the AP endonuclease assay. One unit of uracil DNA glycosylase activity was defined as the enzyme activity required for the release of 1 pmole of uracil from [5- H]uracil-containing salmon sperm DNA under standard reaction conditions. Thin-layer and paper chromatographic systems System A. Whatman No. 1 filter paper developed by descending chromatography for 15 cm with isopropanol/HC1/water (170:41:39 by vol.). Fractions of 0.5 or 1 cm were cut and scintillation counted. System B. Silica gel G60 TLC (Merck) developed for 14 cm with water-saturated ethyl acetate/n-propanol (4 : 1, by vol.). The plates were

processed as described (Reynolds et al., 1981), 0.5-cm fractions were eluted into water and scintillation counted. System C. Whatman No. 1 filter paper developed by descending chromatography for 17 cm with ammonium sulfate-saturated water/1 M sodium acetate (80 : 18 by vol.). Fractions of 0.5 or 1 cm were cut and scintillation counted. Both tritium-labelled and unlabelled uracil and deoxyuridine, and unlabelled deoxyuridine-5'monophosphate were employed as markers.

Purification of AP endonuclease All steps were performed at 4 ° C. The conversion of depurinated supercoiled pAT153 to open circular form was the principal assay employed during purification. To increase protein concentration at any given step, fractions were dialyzed against the appropriate buffer plus 10% PEG 6000. Cells (20 g wet weight) were washed in buffer A (50 mM Tris-HC1, pH 7.6, 10 mM EDTA, 0.1 M NaC1) and resuspended in 30 ml of buffer A containing 0.15 mM phenylmethylsulfonylfluoride before passage through a French press at 30000 psi. Particulate debris was removed by centrifugation. DNA was precipitated by addition of streptomycin sulfate to 1% (w/v) and removed by centrifugation to yield Fraction 1. The 43-75% ammonium sulfate cut of Fraction 1 contained 40% of total protein and 90% of the AP endonuclease activity (Fraction 2). Fraction 2 was dissolved in buffer B (20 mM Tris-HC1, pH 7.6, 2 mM EDTA), dialysed against buffer B, and loaded onto a DEAE-Sephacel (Pharmacia) column (10 cm × 2.6 cm) and equilibrated with buffer B. A step gradient was employed, eluting AP endonuclease activity at 0.3 M NaC1 in buffer B, and then dialyzed against buffer B containing 0.2 M NaC1 and 5% glycerol (by vol.). The resulting fraction (Fraction 3) was loaded onto a gel matrix Sephacryl-200 (Pharmacia) column (70 cm × 1.6 cm), and eluted with buffer B at a flow rate of 5.4 ml/h. Fractions were collected in tubes containing 500 /~g/ml bovine serum albumen (BSA), which stabilized enzymatic activity. The molecular weight (MW) of the AP endonuclease was estimated during the Sephacryl-200 fractionation according to the manufacturer's instructions. The standards were ribonuclease A (MW 13700), carbonic

266

anhydrase (MW 29 200), ovalbumin (MW 45 000), and BSA (MW 66 000). Fractions containing AP endonuclease activity were pooled and dialyzed against buffer C (50 mM NaC1, 10 mM Na phosphate, pH 7.0, 1 mM EDTA, 5% glycerol) to give Fraction 4. Fraction 4 was loaded onto a hydroxylapatite (HT grade, Bio-rad) column (10 cm x 2.6 cm), equilibrated with buffer C, and developed with a linear 10-300 mM phosphate gradient in buffer C. AP endonuclease activity eluted over the range 80-130 mM phosphate with peak activity eluting at 103 mM phosphate. Fractions were collected into tubes containing 500 /~g/ml BSA. Fractions containing 95% of eluted AP endonuclease activity were pooled and dialysed against buffer B with 5% glycerol (by vol.) to give Fraction 5. Fraction 5 was loaded onto a DEAE-Sephacel column (10 cm x 2.6 cm) and developed using 400 ml of a 0-300 mM NaC1 linear gradient in buffer B plus 5% glycerol (by vol.). Fractions were collected into tubes containing 500 /~g/ml BSA. AP endonuclease eluted between 155 and 175 mM NaC1, and the fractions were pooled and dialyzed against buffer B containing 50 mM NaC1. This fraction was diluted with an equal volume of glycerol to give Fraction 6. Fraction 6 was used to characterize the AP endonuclease. Protein concentrations were assayed using the Bio-Rad protein assay kit.

Preparation of extract for detection of uracil DNA glycosylase All steps were performed at 4 ° C. Cells (9 g wet weight) were washed in buffer A, resuspended in 25 ml of the same solution, and the suspension sonicated. Particulate debris was removed by centrifugation. DNA was precipitated by addition of streptomycin sulfate to 1% (w/v) and removed by centrifugation, yielding Fraction 1. The 2-43%, 43-75%, and 75-90% ammonium sulfate cuts of Fraction 1 were dissolved in buffer B, and dialyzed against buffer B containing 5 % glycerol (by vol.). Uracil DNA glycosylase activity was detected in both the 2-43% and 43-75% ammonium sulfate fractions with more activity in the latter. While the activity in the 43-75% fraction was stable upon storage at 4°C, that in the 2-43% fraction was not, losing about 50% of its activity over 5 days. The 43-75% fraction (Fraction 2) was

used for characterization of the uracil DNA glycosylase activity. Results

Transformation of D. radiodurans with uracil-containing DNA D. radiodurans strain 78 is defective in the mtcA gene, which encodes UV endonuclease-a. 1

2

3

4

Form 2 Form 3

Form 1

Fig. 1. Degradation of uracil-containing pUE58 by D. radiodurans crude extract. 5 t~l of crude extract was incubated with 1 btg pUE58 in 20 m M T r i s - H C l , p H 7.0, 10 m M EDTA, 50 m M NaC1 in a total volume of 20 ~1 at 3 7 ° C for 15 min. The reaction was terminated as described in Materials and methods for AP endonuclease assay No. 1, subjected to agarose gel electrophoresis, and stained with ethidium bromide. Form 1 indicates supercoiled plasmid, F o r m 2 open circular plasmid, and Form 3 linear plasmid. Lane 1 : p U E 5 8 without uracil, no crude extract; Lane 2 : p U E 5 8 without uracil, 5 /~1 crude extract. The smear at the bottom of the gel in Lanes 2 and 4 is nucleic acids in the crude extract. Lane 3 : p U E 5 8 with uracil, no crude extract. Lane 4 : p U E 5 8 with uracil, 5 ~1 crude extract.

267 One of the properties of mtcA strains is a marked increase in sensitivity to MMC. Plasmid pUE58 is a 5.6 kb EcoRI fragment of mtcA + chromosomal D N A cloned into the EcoRI site of pAT153 (A1Bakri et al., 1985). Transformation of D. radiodurans strain 78 recipients with pUE58 results in chromosomal recombinants that are resistant to MMC. Using the transformation protocol described in Materials and methods, MMC R transformants were obtained at a frequency of I × 10 -3. When pUE58 was grown in E. coli CJ236 (dut ung), permitting incorporation of uracil into I~UE58 D N A (Taylor and Weiss, 1982), it was consistently found that transformation efficiency was reduced to 1 × 10-4. Plasmid transformation of E. coli D H 5 a with uracil-containing pUE58 was also diminished, while plasmid transformation of E. coil CJ236 ( dut ung) with uracil-containing pUE58 showed no diminution in transformation efficiency, as expected. Cell free crude extract from D. radiodurans was incubated in the presence of E D T A with pUE58 that had been harvested from either E. coli D H 5 a or CJ236 (dut ung) (Fig. 1). pUE58 harvested from CJ236 (dut ung) was severely degraded by crude extract while plasmid harvested from D H 5 a was not (Fig. 1). Although a number of mechanisms can be advanced, this observation is compatible with the presence of a cation-independent

uracil D N A glycosylase and an AP endonuclease in the crude extract.

Purification of AP endonuclease AP endonuclease was purified as outlined in Materials and methods, and summarized in Table 1. The molecular weight of the AP endonuclease was 34 500, as assessed during purification on the Sephacryl-200 gel matrix column. Aliquots from each purification step were compared by 12% SDS-polyacrylamide gel electrophoresis, which showed a progressive decrease in the number of bands visible by silver staining. Fraction 6 contained only 11 bands in addition to BSA. Two of these bands were near 34 500 dalton (not shown).

AP endonuclease assayed by conversion of supercoiled pA T153 to open circular form 1 /tl of Fraction 6, containing 40 units of AP endonuclease, was incubated with 1 /~g supercoiled pAT153 and 1 m M E D T A using the standard reaction conditions for times up to 6 h. The pAT153 was untreated, depurinated, alkylated, or deaminated to contain uracil, as described in Materials and methods. Only depurinated D N A was relaxed to open circular form, while alkylated, normal, and uracil-containing pAT153 were unaffected. Using the same assay, the effects of 10 mM Mg 2+, Mn 2+, Cu 2+, or Zn 2+ on enzyme activity

TABLE 1 PURIFICATIONOF AP ENDONUCLEASE Fraction

Stage

Total protein a (mg)

Total enzymeb (units x 10- 3)

1

Crude extract

600

1050

1850

2

Ammonium sulphate (43-70% cut)

138.2

910

6 600

86.6

3

1st DEAE-sephacel (step gradient)

520

15700

49.5

4

Sephacryl-200

-

312

-

29.7

5

Hydroxylapatite

-

201

-

19.1

6

2nd DEAE-sephacel (linear gradient)

-

80

-

7.6

33.28

Specific activity a (units mg- 1)

Yield (%) 100

a The total protein and specific activity were not calculated for the final three stages because fractions were collected in tubes containing 500/tg/ml bovine serum albumin. b One unit is defined as activity required to convert 1 /~g of depurinated supercoiled plasmid in 30 min at 37 °C to open circular form, as described in Materials and methods.

268

were determined. Once again, only depurinated D N A was cleaved. The rate of cleavage of depurinated D N A was unchanged by the presence of either 10 m M Mg 2+ or Mn 2+, but was completely inhibited by 10 m M Cu 2÷ or Zn 2÷.

AP endonuclease assayed by release of label from E. coli chromosomal DNA Fraction 6 was assayed for release by acid-soluble label from E. coli chromosomal D N A that contained [Me-3H]thymine in the presence of 0.2 m M EDTA, as described in Materials and methods and Table 2. Fraction 6 had no detactable activity towards normal, UV-irradiated, or alkylated D N A (Table 2). Using alkylated D N A that had been depurinated, Fraction 6 released 62% of acid-solubilizable label over a 30-min incubation, calculated as the fraction of acid-soluble label released by N a O H (Table 2). The presence of 10 m M Cu 2÷ or Zn 2÷ completely inhibited AP endonuclease activity. The addition of 5 m M Mg 2+ plus 5 m M Mn z ÷ to the enzyme reaction mixtures produced results identical to those shown in Table 2. The addition of either Mg 2÷ or Mn 2÷ was inhibitory at concentrations in excess of 10 mM. When Fraction 1 (crude) was added to a reaction after both Fraction 6 and divalent cations had already been added, the D N A underwent severe degradation, indicating that Fraction 6 did not contain a nuclease inhibitor. Fraction 6 does not contain an exonuclease activity in the presence or absence of divalent cations, as almost no acidsoluble label was generated from normal, methylated, or UV-irradiated DNA, even after extended incubation (6 h) with 80 units. The AP endonuclease does not recognize all alkali-labile sites in alkylated-depurinated D N A , since an extended incubation (6 h) with enzyme released only 80% of the acid-soluble radioactivity released by N a O H treatment (not shown). The AP endonuclease had optimal activity at NaC1 concentrations between 0.1 and 0.2 M, and p H 7.5. The enzyme is active over the range 0-0.4 M NaC1, and p H 6.0-8.5 (Fig. 2). At 60 ° C the half-life of AP endonuclease activity was 2.5 min (not shown).

Specificity of uracil DNA glycosylase Varying amounts of Fraction 2 were incubated in the presence of E D T A under standard condi-

tions with 0.3 /zg salmon sperm D N A that contained either [Me-3H]thymine or [5-3H]uracil (6 × 104 cpm; specific activity of both substrates 2 × 105 cpm/~tg). Acid-soluble label was detected only if the substrate-DNA contained uracil. A vast excess of extract (5 /zl undiluted, under standard conditions sufficient to release 40 nmoles of uracil-equivalent to 1.1 × 107 cpm) released no

>

~ - - 10 o

•

i

,

i

•

i

•

i

•

w

15 'ID Q r~

== _e

30-

lO

20-

10o 0.0

0.2

0.4

NaCI [MI

0.6

5

6

7

8

9

pH

Fig. 2. NaCI and pH dependence of AP endonuclease and uracil DNA glycosylase activities. Upper left: 80 units AP endonuclease (Fraction 6), 1 vg alkylated-depurinated E. coli chromosomal DNA, and either 7.5 mM Na citrate, pH 7.0, 0.2 mM EDTA, and NaC1 ranging from 75 mM to 775 mM (closed circles) or 10 mM Tris-HCl, pH 7.0, 1 mM EDTA and 0 or 50 mM NaCI (open circles) in a total volume of 40 #1 incubated for 20 rain at 37 o C. Acid-soluble radioactivity was determined as described in Materials and methods. Upper right: A total reaction volume of 40 #1 contained 80 units of AP endonuclease (Fraction 6), 1 /*g alkylated-depurinated E. coli DNA, 100 mM NaC1, 0.2 mM EDTA and either 25 mM Na-citrate/Na-phosphate (pH 4.2 to 6.0; closed circles), or 25 mM Tris-HCl (pH 6.5-9.0; open circles). The reaction was incubated for 20 rain at 37 o C, and radioactivity in the acidsoluble fraction determined. Lower left: 20 units of uracil D N A glycosylase (Fraction 2), 0.18 #g of [5-3H]uracil-contain ing salmon sperm D N A (36000 cpm), 15 mM Tris-HC1, pH 7.6, 5.5 mM EDTA, and varying amounts of NaCI were incubated in a total reaction volume of 10 #1 for 15 rain at 37 o C. Percent uracil released was determined as radioactivity in the acid soluble fraction. Lower right: 32 units of uracil D N A glycosylase (Fraction 2), 0.11 #g of [5-3H]uracil-contain ing salmon sperm DNA (22000 cpm), 2 mM EDTA, and either 10 mM MOPS (pH 5-8.5; closed circles) or 10 mM Tris-HCI (pH 6.5-9; open circles) in a total reaction volume of 14 #1 was incubated for 15 rain at 30 o C. Percent uracil released was determined as radioactivity in the acid-soluble fraction.

269 TABLE 2 ACTIVITY OF THE AP ENDONUCLEASE ON DIFFERENT SUBSTRATES Substrate DNA a

Assay component b

cpm released

Corrected cpm value

pmoles nucleotide released

UT UT UT

NaOH Fraction 6

25 38 35

0 13 10

0 4 3

UV UV UV

NaOH Fraction 6

33 51 34

0 18 1

0 5 0

MMS MMS MMS

NaOH Fraction 6

26 98 65

0 72 39

0 22 12

AP AP AP

NaOH Fraction 6

38 2 854 1780

0 2 816 1742

0 853 528

1 /~g of E. coli chromosomal DNA containing [Me-3H]thymine (104 cpm/#g). UT, untreated; UV, UV-irradiated DNA (600 j/m2); MMS, alkylated by treatment with MMS as described in Materials and methods; AP, alkylated DNA depurinated as described in Materials and methods. b - , no treatment. NaOH, 0.4 M NaOH, 37°C for 30 rain as described in Materials and methods; Fraction 6, 160 units of AP endonuclease per assay; incubations with Fraction 6 were for 30 rain at 37 o C, in SSC, pH 7.0, 0.2 mM EDTA. a

acid-soluble label above background (23 cpm) from [Me-3H]thymine-containing DNA. The absence of acid-soluble material resulting from in-

A

cubation of Fraction 2 with [Me-3H]thymine-con raining DNA indicates a lack of exonuclease activity in Fraction 2 in the presence of EDTA.

B

u__

"°2-"

c

30,

2o, O 10,

0

-

I

10

20

-

,

10

--

---ir'

20

10

20

30

FRACTION

Fig. 3. Degradation products of uracil-containing DNA and deoxyuridine. (A) Standard reaction conditions using [5-3H]uracil-labelled salmon sperm DNA for substrate, as described in Materials and methods. Chromatography in System A. Black bars indicate positions of uracil and deoxyuridine markers. (B) Triangles: Standard reaction conditions using [5-3H]uracil-labelled salmon sperm DNA as substrate. Chromatography in System B. Note that the relative mobility of the uracil and uridine markers is reversed with respect to System A. Solid squares: Standard reaction conditions, except for omission of uracil-containing DNA and addition of 0.3 pmoles deoxy[6-3H]uridine (6000 cpm) instead. Chromatography in System B. Open squares: Standard reaction conditions, except for omission of uracil-containing DNA, and addition of 0.3 pmoles deoxy[6-3H]uridine and sodium phosphate to a final concentration of 10 mM. Chromatography in System B. (C) Standard reaction conditions using [5-3H]uracil-labelled salmon sperm DNA as substrate. Chromatography in System C. In System C deoxyuridine and uracil comigrate (unlike systems A and B), but dUMP has greater mobility.

270 The nature of the label released from D N A that contained uracil was found to be uracil itself, without detectable deoxyuridine or deoxyuridine5'-monophosphate (Fig. 3). Thymidine and uridine phosphorylase in the presence of inorganic phosphate degrades deoxyuridine to yield uracil and deoxyribose-l-phosphate (O'Donovan and Neuhard, 1970). We tested for the presence of this activity under our reaction conditions, since free uracil could have arisen by cleavage of deoxyuridine that had been liberated from DNA by other activities. The absence of these activities under our conditions was demonstrated by incubation of 0.3 pmoles deoxy[6-3H]uridine with an amount of extract sufficient to release 16 nmoles of uracil from uracil-labelled DNA. In the absence of phosphate (our usual incubation condition) no [6-3H]uridine was converted to uracil, while in the presence of 10 mM sodium phosphate about 10% of the [63H]uridine was converted to uracil (Fig. 3B). The presence of 10 mM sodium phosphate had no effect on the release of uracil from uracil-containing salmon sperm DNA (not shown). The optimal reaction conditions for uracil DNA glycosylase activity were 50 mM NaC1 and p H 7.5. The range for activity was 0-0.1 M NaC1 and pH 6.5 to > 9.0 (Fig. 2). At 6 0 ° C the half-life of uracil DNA glycosylase activity was 1.2 min (not shown). Discussion

purified beyond ammonium sulfate fractionation (Fraction 2). It was active in the presence of EDTA, removing [5-3H]uracil from DNA, without discernible formation of deoxyuridine or deoxyuridine-5'-monophosphate. In the presence of EDTA, Fraction 2 released no acid soluble label from D N A containing [Me-3H]thymine, indicating an absence of nuclease activities under these conditions. Both enzyme activities were optimal at pH 7.5. The optimal NaC1 concentrations for AP endonuclease and uracil DNA glycosylase activities differed, being 150 mM and 50 mM, respectively. The half-life of activity when enzyme preparation was heated to 60 ° C prior to assay was 2.5 min for AP endonuclease and 1.2 min for uracil D N A glycosylase. As purified AP endonuclease (Fraction 6) had no activity towards uracil containing-DNA, it may be concluded that the two activities are not physically associated. The quantities of AP endonuclease and uracil D N A glycosylase in D. radiodurans do not appear to vary greatly from E. coli. We estimate that the E. coli crude extract specific activity of the AP endonuclease of exonuclease III, the major AP endonuclease of E. coli, is 180% that of the crude extract specific activity of the AP endonuclease studied here (Verly, 1981). We calculate that the uracil D N A glycosylase specific activity in E. coli crude extract is about 56% of the uracil DNA glycosylase specific activity we detected in crude extract of D. radiodurans (Lindahl et al., 1977).

Properties of A P endonuclease and uracil DNA glycosylase The AP endonuclease was purified (Fraction 6), but not to homogeneity. The estimated molecular weight is 34.5 kD, a size commensurate with AP endonucleases derived from other sources (Weiss, 1987). Fraction 6 had no discernible activity towards normal, alkylated, uracil-containing, or UV-irradiated DNA. The only activity detected was towards depurinated DNA, either pAT153 depurinated by heating, or E. coli chromosomal DNA, depurinated by alkylation followed by heat. The enzyme was active in the presence of EDTA, and divalent cations did not increase activity. Either in the presence or absence of divalent cations Fraction 6 had no detectable exonuclease activity. The uracil DNA glycosylase was not

Base excision repair in D. radiodurans? D. radiodurans has been convincingly demonstrated to be immutable by some DNA damaging agents: UV (254 nm) radiation, ionizing radiation, and mitomycin C (Sweet and Moseley, 1974, 1976; Kerszman, 1975; Tempest and Moseley, 1982). In all these cases this immutability (mutants/survivor) has been shown not only for low exposures, but also for exposures that reduce survival by at least 2 or 3 logs. It has also been shown that damage produced by these agents is repaired by nucleotide excision repair or recombinational repair in D. radiodurans (Moseley, 1983; Moseley and Evans, 1983; Evans and Moseley, 1983, 1985, 1988). On the other hand, several D N A damaging agents are known to be mutagenic in D. radio-

271

durans, although to a lesser degree than in E. coll. These include the alkylating agents N-methyl-N'nitro-N-nitrosoguanidine (MNNG), fl-propiolactone and ethyl methanesulfonate (Sweet and Moseley, 1976). Of these, the most potent mutagen is MNNG, aJmost as effective in D. radiodurans as it is in E. coli (Sweet and Moseley, 1974, 1976). According to the E. coli model, many (but not all) forms of base damage caused by these alkylating agents would be corrected by base excision repair. In addition to mutability by alkylating agents, D. radiodurans undergoes spontaneous mutation at a frequency comparable with E. coli: to streptomycin resistance at ca. 1 x 10 -6 per generation (Kerszman, 1973), rifampin resistance at ca. 5 X 10-8 per generation (Tempest and Moseley, 1982), and to trimethoprim resistance at ca. 1 x 10-5 per generation (Sweet and Moseley, 1974, 1976). Spontaneous mutagenesis could be due to misincorporation, or spontaneous base damage. Like alkylation damage, a variety of spontaneous base damages would be expected to be corrected by base-excision repair initiated by DNA glycosylases, or, in the case of spontaneous base loss, excision repair initiated by an AP endonuclease. While the nature of the repair pathways for alkylation damage and spontaneous base damage remain uncertain in D. radiodurans, the observations made here on the presence of uracil DNAglycosylase and AP endonuclease suggests that base excision repair does indeed occur. Why some damage is repaired in an error-proof fashion, apparently by nucleotide excision repair and recombination functions, while other lesions, probably those corrected by base-excision repair are mutagenic, remains to be determined.

Acknowledgement This work was supported by USPHS grant GM 39933.

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