[29] Purification and properties of Escherichia coli endodeoxyribonuclease V

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residual activity was inhibited by 2 mM EDTA. Manganese ions could partially replace Mg2+; its optimal concentration was 1 raM, and at this concentration the activity was 50% of that observed with 3 mM Mg~÷. Stability. Fraction VI has been stored at 0° for a period of 3 months with no loss in activity. The stability of fraction VII has not been assessed. Additional Substrates. Fraction VI contains no nonspecific endonucleolytic activity when tested in 4000-fold excess on ~bX174 RFI DNA. Exonuclease activity (as determined by release of acid-soluble material) was not observed when either nicked 6X174 DNA or single-stranded E. coli DNA were used as substrates. Phosphomonoesterase activity was undetectable.

[29] P u r i f i c a t i o n a n d P r o p e r t i e s o f Escherichia coli Endodeoxyribonuclease V

By BRUCE DEMPLE, FREDERICKT. GATES III, and STUART LINN

Assay I

Principle. This assay measures the conversion of phage fd single stranded, circular D N A to a linear form which is hydrolyzed to acidsoluble products by Escherichia coli exonuclease I.

Reagents 1. The reaction mixture contains in 150/zl 67 mM glycine-NaOH, pH 9.5 25 mM KC1 10 mM MgCI~ 27/~M all-labeled fd DNA 1 (4 nmole total DNA-nucleotide; 3 to 6 × 10~ cpm/nmole) 0.85 to 2 units exonuclease I s 1 Prepared as described by P. J. Goldmark and S. Linn, Proc. Natl. Acad. Sci. U.S.A. 67, 434 (1970). Unless otherwise noted, all DNA concentrations are expressed as DNA nucleotide residues. I. R. Lehman and A. L. Nussbaum, J. Biol. Chem. 239, 2628 (1964). One unit of exonuclease I hydrolyzes 10 nmoles of DNA nucleotide to acid-soluble material in 30 rain at 37°. The amount of exonuclease I added to the endonuclease V reaction mixture should be sufficient to render at least 4 nmoles of DNA nucleotide acid soluble under the conditions of the assay, while introducing a minimal number of endonucleolytic cleavages into the fd DNA. For our preparations of exonuclease, this is 0.85 to 2 units.

METHODS IN ENZYMOLOGY, VOL. 65

Copyright © 1980 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181965-5

ENDO V OF E. coli

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225

Bovine serum albumin, 10 mg/ml 3. Trichloroacetic acid, 7% (w/v) 4. Aqueous scintillation fluor: 9.1 g of 2,5-diphenyloxazole (PPO) and 0.61 g of 1,4-bis[2-(5-phenyloxazoyl)]benzene (POPOP) are mixed for 1 hr with 2140 ml toluene. 1250 ml Triton X-100 is then added and mixing is continued for 5 hr .

Procedure. An appropriate amount of endonuclease is added, then the reaction mixture is incubated for 30 rain at 37°C. It is then chilled on ice, and 0.05 ml of bovine serum albumin (10 mg/ml) and 0.25 ml of 7% TCA are added with mixing following each addition. After 5 min at 0°, the precipitate is removed by centrifugation at 10,000 g for 5 min; a 300-~1 aliquot of the resultant supernatant is mixed with 3.3 ml aqueous fluor in a "minivial," and the radioactivity determined in a scintillation counter. (If a standard 20-ml vial is used, the aliquot should be brought to 0.9 ml and 10 ml of the fluor should be utilized.) Calculations. One unit of endonuclease activity is defined as the amount which produces 1 nmole of acid-soluble product during the 30 min incubation. The assay is linear over the range of 0.15 to 1.5 units. A blank value of 0.3 nmole or less of acid-soluble material is observed in the absence of endonuclease, due to linear fd DNA and/or contamination of the exonuclease I by endonuclease. The number of endonucleolytic cleavages can be estimated by determining the number of fd DNA molecules opened during the incubation. An fd DNA molecule contains 6389 nucleotides, so the acid-solubilization of 1 nmole of DNA is taken to be due to the conversion of 0.157 pmole of DNA molecules from the circular to the linear form. Since only one endonucleolytic cleavage is required for each such conversion, a minimum number of such cleavages is estimated unless one utilizes a Poisson correction (see assay II).

Assay II

Principle. This assay measures the conversion of closed circular duplex DNA from phage PM2 to the nicked form which can be selectively denatured and bound to nitrocellulose filters. Reagents 1. The reaction mixture contains in 50/~1 67 mM Tris-HCl, pH 8.2 25 mM KC1 10 mM MgCI2

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2. 3. 4. 5. 6. 7. 8. 9.

I0.

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20-100/zM 3H-labeled PM2 DNA 3 (1-5 nmoles total DNA nucleotide; 3-10 × 10a cpm/nmole 2.5 mM EDTA, pH 8.0-0.01% sodium dodecyl sulfate CHCls : n-octanol (9 : 1) 0.3 M potassium phosphate adjusted to pH 12.3 as measured at 0.3 M with 5 N KOH 1.0 M potassium phosphate adjusted to pH 4.0 as measured at 1.0 M with 5 N HaPO4 5 M NaC1 50 mM Tris-HCl, pH 8.2-1 M NaC1 0.3 M NaC1-0.03 M sodium citrate Nitrocellulose filters (Schleicher and Schuell type BA85, 0.45/xm pore size), equilibrated with the Tris-NaC1 solution at least 12 hr before use Nonaqueous scintillation fluor: 24 g of 2,5-diphenyloxazole (PPO) and 0.6 g of 1,4-bis[2-(5-phenyloxazoyl)]benzene (POPOP) are mixed for 1 hr with 6 liters of toluene

Procedure. An appropriate amount of endonuclease V is added, then the reaction mixture is incubated for 30 min at 37°. The reaction mixture is chilled on ice, then 0.5 ml EDTA-SDS solution is added. For fractions I-V, the mixture is then extracted at room temperature with an equal volume of chloroform-octanol solution. (For more purified fractions the extraction step is unnecessary.) A 0.2-ml aliquot of the supernatant is mixed with 0.1 ml water and counted for total radioactivity in 3.3 ml aqueous fluor (see Assay I). Another 0.2-ml aliquot is added to 0.2 ml of 0.3 M potassium phosphate, pH 12.3, at room temperature. After 2 min, 0.15 ml of 1.0 M potassium phosphate, pH 4.0, is added, followed by 0.14 ml of 5 M NaCI and 5.0 ml of Tris-NaCl solution. The sample is then passed through a nitrocellulose filter. The filter is washed with 5 ml of the NaCl-sodium citrate solution once before and once after the sample has been passed over it. The filter is then dried under an infrared lamp, and radioactivity determined by counting in 5 ml of nonaqueous scintillation fiuor.

Calculations. The number of nicks formed during the incubation with enzyme can be calculated by assuming that the target sites are distributed among the DNA molecules according to a Poisson distribution and that all sites have an equal probability of becoming nicked. The equation U = e-~' can then be used to determine the average number of nicks per molecule, /z, where U is the fraction of molecules not nicked, i.e., the fraction not a Prepared by the method of R. T. Espejo and E. S. Canelo [Virology 34, 738 (1968)] as modified by U. Kiihnlein, E. E. Penhoet, and S. Linn [Proc. Natl. Acad. Sci. U.S.A. 73, 1169 (1976)].

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TABLE I PURIFICATIONE. coil ENDONUCLEASEVa

Fractionb I. Crude extract II. Streptomycin supernatant III. Ammonium sulfate precipitate IV. Potassium phosphate precipitate V. DEAE-cellulose VI. Phosphocellulose VII. CM-Sephadex concentrate VIII. Glycerol gradient

Volume (ml)

Protein (mg)

Enzyme activity (units × 10-3)

Specific activity (units/rag)

453 482

5890 5300

398 304

69 57

222

4880

357

73

80

1400

302

216

455 80 4

114 2.24 0.12

69.6 38.0 6.92

611 17,000 57,000

28

<0.03

6.92

>230,000

a Activity was measured by assay I. b Protein in Fractions I through VI was determined by the method of O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). Protein in Fractions VII and VIII was determined by the microassay system of M. M. Bradford, Anal. Biochem. 72, 248 (1976). Bovine serum albumin was used as the standard in all protein determinations. b o u n d to t h e n i t r o c e l l u l o s e filter, (The t o t a l D N A is o b t a i n e d f r o m t h e first a l i q u o t r e m o v e d f r o m t h e r e a c t i o n m i x t u r e . ) It s h o u l d b e n o t e d t h a t U is a l w a y s ~< 1 a n d t h a t / z i n c r e a s e s e x p o n e n t i a l l y as U a p p r o a c h e s 0. T h e r e fore the most accurate determinations of enzyme activity are obtained w h e n less t h a n 1 n i c k p e r m o l e c u l e is f o r m e d , i . e . , U / > 37%. O f t h e D N A m o l e c u l e s in t h e p r e p a r a t i o n , 5 - 1 0 % a r e n i c k e d a f t e r i n c u b a t i o n w i t h o u t e n z y m e ; this v a l u e is m e a s u r e d a n d u s e d to c o r r e c t t h e c a l c u l a t i o n s . Purification Procedure U n l e s s o t h e r w i s e n o t e d , all o p e r a t i o n s a r e c a r d e d o u t at 0o-4 ° . Crude extract. Escherichia coli s t r a i n JC4583 ( a n e n d o n u c l e a s e 1-deficient K12 s t r a i n ) is g r o w n to m i d - o r l a t e - l o g p h a s e in L b r o t h 4 at 37 ° w i t h f o r c e d a e r a t i o n , t h e n h a r v e s t e d b y c e n t r i f u g a t i o n at 4000 g f o r 5 rain. T h e cell p a s t e c a n b e s t o r e d for u p to 3 m o n t h s at - 2 0 ° a f t e r q u i c k f r e e z i n g in l i q u i d n i t r o g e n . I n o n e s u c h p r e p a r a t i o n (Table I), 80 g o f cell p a s t e w e r e s u s p e n d e d in 50 m M g l y c y l g l y c i n e - N a O H , p H 7.0---1 m M p o t a s s i u m p h o s p h a t e , p H 6 . 8 ~ 0 . 1 m M d i t h i o t h r e i t o l (buffer A ) to a t o t a l 4 G. Bertani and J. J. Weigle, J. Bacteriol. 65, ll3 (1953).

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volume of 200 ml. The cells were then broken by five, 45-sec pulses from a Branson Sonifier at 110 W, the temperature being maintained between 2° and 10° by an ice-salt bath. After centrifugation at 25,000g for 20 min, the supernatant was diluted with buffer A to an absorbance of 200 at 260 nm (fraction I). Streptomycin Precipitation. To fraction I was added slowly with stirring 0.085 volume of 5% streptomycin sulfate (Schwarz-Mann) in buffer A. After 30 min, the precipitate was removed by centrifugation at 25,000g for 25 min and discarded (fraction II). Ammonium Sulfate Precipitation. To each 100 ml of fraction II were added with stirring 37.9 g enzyme grade ammonium sulfate (SchwarzMann). The pH was maintained at 7.0 by the addition of 7 N NI-I4OH. After 30 min, the precipitate was collected by centrifugation at 25,000 g for 15 min, and resuspended in buffer A to an absorbance of 350 at 260 nm (fraction III). Dibasic Potassium Phosphate Precipitation. To fraction III was added slowly with stirring 0.155 volume o f 4 M K2HPO4. After 10 min on ice, the precipitate was removed by centrifugation for 10 min at 27,000 g. A second aliquot of 4 M K2HPO4 (0.41 volume based on fraction III) was added slowly with stirring. After 30 min the precipitate was collected by centrifugation for 25 min at 27,000g and resuspended in a minimal volume of buffer A. This suspension was then dialyzed for 12 hr against 40 volumes of buffer A (fraction IV). DEAE-Cellulose Chromatography. A column containing 450 ml of packed resin 5 was equilibrated with buffer A. Fraction IV was applied to the column at 1.5 ml/min. The column was washed with 300 ml of buffer A, then a linear gradient from 0 to 0.4 M KC1 of buffer A (4 liters total) was applied at 1.5 ml/min. Twenty milliliter fractions were collected into tubes containing 0.8 ml of 0.5 M (NI-I4)2SO4-0.5 M potassium phosphate (pH 7.0). The active fractions (0.11 to 0.17 M KCI) were pooled and dialyzed for 12 hr against 10 volumes of buffer B (30 mM potassium phosphate, pH 6.8--0.5 mM dithiothreitol) (fraction V). Phosphocellulose Chromatography. A column containing 150 ml of packed resin 5 was equilibrated with buffer B. Fraction V was then applied at a rate of 1.5 ml/min. The column was washed with 90 ml of buffer B, then a linear gradient from 0 to 0.6 M KCI in buffer B (1.2 liters total) was applied at 1 ml/min. Ten milliliter fractions were collected into polypropylene tubes. The active fractions (0.33 to 0.36 M KC1) were pooled and dialyzed for 10 hr against 50 volumes of buffer B (fraction VI). CM25-Sephadex Chromatography. A column containing 30 ml of packed CM-Sephadex 5 was equilibrated with buffer B. Fraction VI was DEAE-cellulose, type 40, is purchased from the Brown Co., Berlin, New Hampshire. Phosphocellulose P-11 is a Whatman product. CM25-Sephadex is from Pharmacia.

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TABLE II RELATIVE RATES OF DIGESTION OF VARIOUS DNAs BY ENDONUCLEASE V a DNA fd Untreated PM2 Relaxed PM2 UV-irradiated PM2 Bisulfite-treated PM2 PM2 exposed to pH 5.2 OsO4-treated PM2 PBS-2

pH 8.2

pH 9.5 30.3

-= 1.0 1.0 4.0

0.7 3.7 7.5 ~ 120

a PM2 DNA was "relaxed" to untwisted form I with E. coil omega protein. PM2 DNA (0.7 to 1.0 m M in 10 mM Tris-HCl, pH 7.5) was irradiated with ultraviolet light in a watch glass on ice for 5 rain with 20 erg/mmZ/sec from a Westinghouse G15T8 Sterilamp and diluted immediately into the reaction mixture. PM2 DNA was treated with sodium bisulfite so as to produce 1-2 uracil residues per DNA molecule as described by Lindahl e t al. 7 DNA was depurinated by treatment at pH 5.2 at 70 ° for 10 min in 0.1 M NaCI-10 m M sodium citrate-10 m M Tris-HCl. This treatment forms roughly 1.5 apurinic sites per PM2 DNA molecule. For OsO4 treatment, 1.2 m M DNA in 25/zl of 10 m M Tris-HC1, pH 7.5, was partially denatured in 100/~1 of 0.3 M potassium phosphate, pH 12.3, at room temperature, then exposed to 15/zl of 1% OsO4 for 30 rain. This treatment produces pyrimidine bases saturated at the 5,6-double bond. The DNA was renatured by the addition of 75/zl of 1 M potassium phosphate, pH 4.0, and 50/~1 of 5 N NaCI and then dialyzed against 10 m M Tris-HCl, pH 7.5. Reactions containing PBS-2 DNA 8 were as for fd DNA, except that I nmole of DNA nucleotide is added and the exonuclease I is omitted.

applied to the column at 0.6 ml/min. The column was washed with 24 ml of buffer B, then a linear gradient from 0 to 0.5 M KC1 in buffer B (400 ml total) was applied at 0.6 ml/min. Six milliliter fractions were collected into polypropylene tubes. The active fractions (0.20 to 0.23 M KCI) were pooled and dialyzed for 10 hr against 100 volumes of 30 mM potassium phosphate, pH 6.8---0.05 mM dithiothreitol (buffer C). The dialyzed material was concentrated by application to a 2-ml phosphocellulose column in a 5-ml polypropylene syringe at 4 ml/hr. After washing with 5 ml of buffer C, the activity was eluted into polypropylene tubes with buffer C containing 0.5 M KC1 (fraction VII). Glycerol Gradient Sedimentation. A 0.10-ml portion of fraction VII was layered onto a 5.2-ml linear glycerol gradient (5 to 20%) in 50 mM potassium phosphate, pH 6.8-10 mM (NI-I4)~SO4-0.05 mM dithiothreitol in a polyallomer tube. After centrifugation for 24 hr at 50,000 rpm in a Spinco SW-50.1 rotor, fractions were collected into polypropylene tubes by dripping from the bottom of the gradient tube. A single, symmetrical peak of activity was observed approximately 40% down the tube (fraction VIII).

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The purified fractions are unstable to freeze-thawing and long-term storage, although they are somewhat stable for several months in 50% glycerol at - 2 0 ° in polypropylene tubes. 6 Endonuclease V activity is also sensitive to dialysis; losses can be minimized by using Spectrapore 1 dialysis membrane (6,000 to 8,000 dalton cutoff). 6 Properties Specificity. The enzyme makes endonucleolytic cleavages in singlestranded DNA, acting at many sites in the fd D N A molecule. 6 Duplex D N A is a substrate o f tenfold lower susceptibility (Table II). 7'6 Relaxation of the PM2 DNA to a nonsupercoiled form, or exposure to uv light, sodium bisulfite (to form a few uracil residues), p H 5.2 (to form apurinic sites), or osmium tetroxide (to form pyrimidines saturated at the 5,6double bond) renders the duplex DNA as much as sevenfold more active as a substrate for endonuclease V 6 (Table II). The e n z y m e has similar relative activities on linear duplex T7 DNA exposed to the various insults .6 The duplex D N A of the Bacillus subtilis bacteriophage, PBS-2, s in which thymine is totally substituted by uracil, is the best substrate. Indeed only this D N A is rendered acid soluble by the purified enzyme. The enzyme does not appear to have an exceptionally high at~inity for uracil in DNA, however, since the PM2 DNA containing a small number of uracil residues introduced by NaHSO3 is cleaved at about the same rate as untreated PM2 DNA (Table II). Effect o f Metals. Endonuclease V exhibits a strong requirement for Mg ~+ and is completely inactive on fd D N A in its absence. Manganese ions can restore some o f the activity on PBS-2 D N A (15% at 2 m M Mn2+), whereas Ca 2+, Zn z+, Co s+, or Fe z+ cannot activate the enzyme. 6 A unique property o f endonuclease V is the strong inhibition by Fe a+ with either fd or PBS-2 DNA. 6'9 Effect o f p H . With fd D N A substrate, endonuclease V exhibits a sharp optimum at pH 9.5. The e n z y m e is also 50-fold more active on duplex substrates at this p H than at p H 7.0. Molecular Size. When sedimented through a glycerol gradient by the

e F. T. Gates and S. Linn, J. Biol. Chem. 252, 1647 (1977). r T. Lindahl, S. Ljungquist, W. Siegert, B. Nyberg, and B. Sperens, J. Biol. Chem. 252, 3286 (1977). 8 PBS-2 DNA is prepared as described by E. C. Friedberg, A. K. Ganesan, and K. Minton [J. ViroL 16~ 315 (1975)], except that 5 mCi of 6-SH-uridine (Schwarz-Mann) are added immediately after infection, and the culture was treated with chloroform and harvested immediately after lysis. R. V. Blackmore and S. Linn, Nucleic Acids Res. 1, 1 (1974).

[30] NONPYRIMIDINE DIMER UV DAMAGE-SPECIFIC ENDONUCLEASE d

231

method of Martin and Ames, 1° endonuclease V exhibits an S2o,w of 2.3. Assuming that the protein is globular, this corresponds to a molecular weight of approximately 20,000. 10 R. G. Martin and B. N. Ames, J. Biol. Chem. 236, 1372 (1961).

[30] P u r i f i c a t i o n a n d P r o p e r t i e s o f a n E n d o n u c l e a s e S p e c i f i c for N o n p y r i m i d i n e D i m e r D a m a g e I n d u c e d b y U l t r a v i o l e t Radiations By SHEIKH RIAZUDDIN

Assay Method

Principle. The assay measures formation of the enzyme-substrate complex prior to incision of the substrate by the endonuclease. E + S ~ES--*

E + P

Reagents 1. Reaction mixture (total volume 0.1 ml) contains Tris-HCl, 10 mM, pH 7.4 2-Mercaptoethanol, 1 mM NaC1, 100 mM Escherichia coli [aH]DNA, 5000 cpm, 250 pmole of mononucleotide equivalent Enzyme, 0.2-1.0 units 2. Saline citrate (300 mM NaC1 + 30 mM sodium citrate)

Procedure. After incubation at 0° for 10 min. the reaction mixture is diluted with 2 ml of cold saline-citrate and filtered immediately through HAWP Millipore filters. The reaction tubes are washed once with 4 ml of cold saline-citrate, and the filters are dried and counted in 2 ml of toluene-containing scintillation fluid. The radioactivity retained on the Millipore filters is taken as a measure of endonuclease binding activity and the nonpyrimidine dimer uv damage specific activity is determined by the difference in binding activities against heavily uv-irradiated (1000 J/m ~) and lightly uv-irradiated (50 J/m 2) DNA substrates. This binding assay is used during the purification procedure to test various fractions for the presence of this enzyme. However, for quantitative purposes, another assay 1 is used. This assay measures the release of 1 s. R. Kushner and L. Grossman, this series, Vol. 21, Part D [15]. METHODS IN ENZYMOLOGY,VOL. 65

Copyright ~) 1980by Academic Press, Inc. All rightsof reproduction in any form reserved. ISBN 0-12-181965-5