ANALYTICAL
BIOCHEMISTRY
Purification
95, 77-81 (1979)
of the Photoreactivating
Enzyme from Yeast’
ROGER M. HERRIOTT Depurtment
of Biochemistry, School of Hygiene 615 North Wolje Street,
and Public Baltimorr.
Health. Murylund
The Johns 21205
Hopkins
University,
Received October 12. 1978 A method of purifying the DNA photolyase (EC 4.1.99.3) from yeast has been developed. The method includes one step which has been included to destroy enzymes that may be responsible for the instability of purified preparations.
unequivocal method. The general procedures for carrying out genetic transformation in this system have been described in detail elsewhere (6,7) but an improved method of making the wild-type cells competent is worth describing. Enzyme substrate. Purified transforming DNA carrying the resistance marker to 2000 pg/ml of streptomycin and capable of transforming 1 to 2 x 10” cells per microgram of DNA taken up by competent cells was diluted in saline-O.02 M PO1, pH 7.2, to a concentration of 2 &ml. Of this solution, 25 ml was placed in a 14-cm sterile petri dish at a distance of 55 cm from a 15-W GE sterilamp. Exposure time was 40 s, during which the dish was gently rotated. This exposure to approximately 1000 ergs/mm* reduced the transforming activity to 10 to 15% of value prior to exposure. This solution kept without change for several months at 5°C in a capped tube. The unit of enzyme activity (Eil~‘).~A unit of photoreactivating enzyme is defined as that quantity which raises the transforming activity of 1 j&ml of 10 to 15% active uvdamaged streptomycin-resistant H. influen?lae DNA at the rate of 1 x lo6 transformants per minute under defined conditions. The
Yeast as a source of the photoreactivating enzyme (DNA photolyase, EC 4.1.99.3) has the advantage over other sources in being readily available commercially in large relatively reproducible quantities. This advantage has been recognized by a number of investigators (l-4). Most of these workers found, however, that the stability of the enzyme decreased as it was purified. This writer, on finding that a variety of modifications in the medium produced no appreciable increase in stability, considered the possibility of a protease which accompanies the photolyase during purification and leads to the latter’s instability. A method of eliminating such a protease without a large loss of photolyase was developed based on Rupert’s finding (5) that the enzyme is protected against inactivation at 66°C by the presence of DNA exposed to ultraviolet light. Quantitative estimates of the stability of preparations from the new procedure have not been made. METHODS Enzyme assay. This assay consists of the initial rate of restoring the activity of uvinactivatedH. influenzae-transforming DNA carrying the genetic marker for high-level resistance to streptomycin. It is a long but
’ Abbreviations used: EU. enzyme unit, DTT. Dithiothreitol: HI, heart infusion; HI*, heart infusion plus 10 pg/ml of hemin and 2 to 4 &ml of DPN: TCA, trichloroacetic acid.
’ This paper is dedicated to the memory of Dr. Alvin Nason. 71
0003.2697/79/070077-05$02.00/O Copyright 6: 1979 by Academx Pre\\. Inc All r,ght\ of reproduct,on ,n itnv frxm re\rr\cd.
78
ROGERM.HERRIOlT
defined conditions include: a medium of 0.15 M NaCl-0.02 M phosphate, pH 7.5-2 mM dithiothreitol (DTT)-I mM EDTA and 5 x lop4 M reduced glutathione. One to two milliliters of enzyme and substrate were placed in a l-cm diameter test tube that was illuminated in a glass-bottom water bath kept at 37°C 1 to 2 cm from three parallel GE F20T 12 BL lamps. Diluent solutions. (a) For the enzyme: 100 &ml of bovine serum albumin-O.15 M saline-0.02 M P04, pH 7.5-2 mM DTT- 1 mM EDTA. (b) Diluent of DNA for assay: 0.15 M NaCl-0.02 M Na, citrate. Assay procedure. (Work in dim or yellow light.) (a) Dilute the enzyme in cold enzyme diluent to about 10 EU/ml and note the dilution factor. (b) Warm 2 ml of enzyme and illuminate at 37°C for 10 min; then chill and keep dark. This is to repair any uv lesions in the DNA that may be in the enzyme preparation. (c) To 1 ml of cold substrate, add 0.04 ml of 0.05 M reduced glutathione and 1 ml of enzyme in b; let stand 30 min cold and dark. (d) Warm tube c at 37°C for 5 min and remove a 0.1-ml sample, which will be diluted serially O.l/lO, then O.l/lO, in DNA diluent. This is the 0 time sample. (e) Turn on the illuminator and allow 5 min to reach a steady output. Place the test tubes with the enzyme-substrate mixture in the illuminated water bath and withdraw 0. I-ml samples after 2, 4, and 8 min of exposure and dilute as in d. (f) Put 0.2 ml of the last dilution of each sample into 1.6 ml of heart infusion, and follow with 0.2 ml of freshly thawed competent H. influenzae. Let stand 30 min at 37°C.
(g) Place 0.5 ml of this transformation mixture onto each of two petri dishes and add, with gentle mixing, 10 ml of warm (43°C) heart infusion*-agar (* means it has had hemin and DPN added to levels of 10 pg and 2 &ml, respectively). When the agar has solidified (15 min) the plates are incubated at 37°C for 1.5 h, and then overlayed with
another 10 ml of heart infusion*-agar containing 100 pg/ml of streptomycin sulfate. When the agar has set, incubate for 10 to 24 h and count the colonies. (In this assay only 50 pg/rnl streptomycin is used although the cells can grow in the presence of 2000 pg/ml.) (h) Control plates to be included: (1) no DNA but competent cells; (2) DNA but no competent cells. Neither control plates should show more than an occasional colony if the stocks are clean. (i) Sum the colony counts of the two plates for each sample and subtract any blanks or controls. Plot the difference in colony count against the time of illumination. The slope of the best straight line through the points times 1 x lo5 (the dilution factor) is now divided by 1 x lo6 to get the number of EU. (j) The number of EU/ml of initial solution is the number of EU found in i times 2x the dilution of enzyme in step 1. (The 2x is due to the dilution with substrate.) EXAMPLE: If the enzyme was diluted IOOOfold (103) and the plot showed a slope of 25 transformations per minute, then the EUlml of original is 25 x 1 x lo5
1 x 106
x 2 x IO3 or 5 x 103.
Preparation of competent (transformable) H. injluenzae. Although this process has
been described previously (6,7) some modifications or details deserve to be mentioned. Some of the stock solutions used in making M-IV solution are not as stable as appeared earlier. Thus, stock No. 22 keeps only a few weeks refrigerated. Stock No. 40 should be made of certified Difco casamino acids and it keeps only 10 days to 2 weeks. The dry casamino acids should be kept in a desiccator and the solution should be filter sterilized. The M-IV solution is best if kept only a week refrigerated. 1. Wild-type (streptomycin-sensitive) Rd H. injluenzae known to yield competent cultures were grown in heart infusion (HI) plus 10 pg/ml of hemin and 2 to 4 p.g/ml of DPN. The complete growth medium is designated
PURIFICATION
OF DNA
HI*. Stocks of such cells at a concentration of 1 x 10g/ml were frozen at -70°C after first adding glycerin to 15% or dimethyl sulfoxide to 20%. 2. A sample of frozen stock was diluted 102, 104. and lo6 in HI and 0.05 ml of each dilution was placed in 5 ml of HI* at 5 PM and rotated at 37°C overnight at about 200 rpm. Also included was a tube containing medium alone (HI*) and one of HI + cells but no hemin or DPN. 3. Early the next morning the two control tubes were clear, indicating that there was no contaminant in the system; 1 ml of the highest dilution showing a turbid culture was introduced into 50 ml of HI* in a 500-ml flask with a 28-mm side tube and rotated at 175 rpm in a New Brunswick water bath at 37°C. When the turbidity, compared to an HI* blank, reached 0.1 o.d. in a Coleman Jr. spectrophotometer set at 650 nm (usually about 2.5 h) the cells were centrifuged in a table model Sorvall SS- 1 centrifuge for 3 min at a powerstat setting of 27. In rapid succession the centrifuge was stopped manually, the supematant was decanted, and the cells were resuspended in 20 ml of warm M-IV; this suspension was centrifuged as before and stopped quickly; the supernatant was decanted away and cells were resuspended in 20 ml of warm M-IV and placed in a 250- to 300-ml Erlenmeyer flask and rotated for 100 min at 125 rpm at 37°C. The cells again were centrifuged at setting 27 and the cells were resuspended in 20 ml of cold HI* containing 15% glycerin or 20% dimethyl sulfoxide. Two to four milliliters of the cells were placed in sterile vials and frozen immediately at -70°C. The competence of cells prepared in this manner was usually greater than 1%. Most of the cells are competent but only 1% get the streptomycinresistance marker. Competence can be measured in several ways but for purposes of this paper the percentage of cells transformed to streptomycin resistance when an excess (1 &ml) of the srrep’ DNA was present (6) has been used. The frozen cells should be thawed quickly
79
PHOTOLYASE
just before use. The cells at -70°C hold their competence for up to 1 month. Microprotein determination. This procedure is for use when the concentration of protein is below 50 pg/ml. The reagents are those of Lowry et al. (8): (A). 10 ml of 2% Na,CO, + 0.1 ml of 1% CuSO, + 0.1 ml of 2.7% NaK tarn-ate; (B). 1 N Folin-Ciocalteu Reagent. To any volume of solution containing 5 to 25 ,ug of protein is added 0.25 vol of cold 50% TCA. Let stand for 10 min; then centrifuge for 10 min; decant carefully: wash 2x with 10% TCA unless you know there are no interferring contaminants such as -SH compounds. Dissolve the precipitate in 0.2 ml of 0.5 N NaOH. Add 1 ml of reagent A and let stand for 10 min; add 0.1 ml of reagent B with mixing and let stand at room temperature for 45 min. Read in l-ml cuvettes in a Beckman spectrophotometer at 700 nm. Run standards and a blank with each determination. RESULTS
The method of preparing the photolyase from yeast is described in Table 1 along with the analytical data indicating the recovery of enzyme activity. The protein content of fraction No. 5 was 6 to 10 mg/ml, so the specific activity (EU/mg of protein) was about 8000, or nearly 500-fold higher than that of a buffer extract of dried yeast. The average yield from several preparations was 42% of the initial activity. Solution No. 5 was stored frozen after the addition of 1 ml of glycerin-or carried on to the affinity chromatography step. Additional
Purijicution
Subsequent purification of the new preparations by affinity chromatography on an uv-exposed DNA cellulose columns ( 11) have not been repeated sufficiently to report the results at this time. Preparations that had no heat treatment but were precipitated by streptomycin sulfate in the presence of
3
4
5
The dialyzed material from No. 3 was mixed with 5 ml of 1.6 mg/ml calf thymus DNA in 0.1 M NaCl that had been exposed to 1 x lo6 ergs/mm* of uv radiation with stirring. (Twenty-five milliliters of DNA on 14-cm petri dish rotated 14 cm from GE Sterilamp for 40 min.) Let DNA + enzyme stand cold and dark for 1 h. Warm to 35°C in a liter beaker. Introduce a strong magnetic stirrer and add rapidly an equal volume of 0.05 M POd2-, pH 7.5-2 mM ME- 1 mM EDTA heated to 92°C. The temperature of the mixture rises to 60°C. Let stand 2 to 3 min; then chill in ice water. Centrifuge at 7K for 10 min. Save the supernatant.
Number 4 $ 2 vol of cold 1 mM ME-EDTA + 1 vol of 1.6% streptomycin sulfate; let stand 3 h or overnight at YC. Centrifuge for 10 min at 7K; discard supematant; dissolve residue in 10 ml of either 0.25 M PO,*-, pH 7.5 or 0.2 M KCI-0.05 M Tris-HCl-1 mM EDTA-5 mM DTT-0.02 M PO,*--5% glycerine, pH 7.5. This latter solvent is used when further purification by affinity chromatography on a DNA column exposed to uv is to follow.
g) was stirred in to bring the AS concentration to discard the supematant and dissolve the residue in mM EDTA. Dialyze this overnight in washed (10) same solvent in a tall jar with stirring. Open the
Supematant No. 0.55 saturated. 206 ml of cold s-in. Visking dialysis bags.
2 + 130 g/liter of solid AS (or 400 Centrifuged at 7K for 20 to 30 mitt; 0.05 M P0,2m, pH 7.5-2 mM ME-l tubing against 6 to 7 liters of the
2
Dried yeast (700 g) mixed slowly with 3.5 liters of warm 0.1 M K,HPO,-5 m&t mercaptoethanol (ME)- 1 mM EDTA in a 6-liter flask. Toluene (22 ml) was added and stirred with a magnetic stirrer at 37°C for 4 h. The solution was chilled by placing the flask in cold water; 735 g of solid ammonium sulfate (AS) was added and stirred slowly until the AS had dissolved. After standing overnight the precipitate was centrifuged at 7K for 0.5 h in a Sorvall 2B centrifuge. The supematant was saved.
No.
1
OFPHOTOLYASE
1
Fresh baker’s yeast was air-dried by crumbling it to small particles on brown wrapping paper and allowing it to stand on the laboratory desk. It was stored at 32°F or lower. This is the procedure of von Lebedev (9) and was used by Rupert (1).
Procedures and materials
PARTIALPURIFICATION
TABLE
10
750
400
2900
Volume
50,000
650
2000
400
Enzyme units per milliliter
5 x 105
5 x 105
Total enzyme units
PURIFICATION
uv-exposed DNA have been chromatographed on such columns after first separating the enzyme from the uv-exposed DNA by ammonium sulfate precipitation. This yielded another 50-fold rise in specific activity. When this was followed by passage through a column of unirradiated DNA more protein was removed with a 2- to 3-fold increase in specific activity. These preparations were unstable perhaps because they were so dilute (3 pg of protein/ml) or because of the hypothetical protease which the present method should reduce. The preparations of highest specific activity (IO5 above an aqueous extract of the yeast) were similar in many ways to those reported by Werbin’s group (4, 18). This purification is encouragingly close to a figure calculated by Harm and Rupert from their photoflash studies (12). There was an absorption maximum at 370 nm in the preparations of high specific activity as well as one at 280 nm. There was also a strong fluorescence emission maximum at 470 nm when exposed to 360-nm radiation. DISCUSSION
81
OF DNA PHOTOLYASE
a small loss in photolyase activity, as expected from Rupert’s earlier studies (5). It remains to be determined if the hoped for stabilization of highly purified preparation has been realized. ACKNOWLEDGMENT The writer acknowledges with gratitude the assistance received in this and related studies by postdoctoral associates Sang Won Park and William Hauswirth, by Margarita Jiminez, and by technical assistants Genny Armilei, Lynne Vique, Robert Union, and Sandra Richter Takai. The competence procedure was examined by Matthew Buechner. This investigation was supported by Public Health Service Research Grant AI-01218 from the NIAID and by Atomic Energy Commission Contract AT(30- 1) I37 I (NYD-1371-58).
REFERENCES 1. Rupert, C. S. (1960) J. Gen. Physiol. 43, 573. 2. Muhammed, A. (1966) J. Biol. Chrm. 241, 516523. 3. Setlow, J. K., Personal communication. 4. Minato, S.. and Werbin, H. (1971) B&hemi.rtrj 10, 4503-4508. 5. Rupert. C. S. (1962)J. Gen. Physiol. 45,725-741. 6. Herriott, R. M., Meyer, E. Y., and Vogt, M. (1970) J. Bacterial. 101, 517-524. 7. Herriott, R. M. (1971) in Chemical Mutagens (Hollaender. A., ed.). pp. 175-212. Plenum Press. New York. 8. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall. R. J. (1951) J. Biol. Chem. 193, 265. 9. von Lebedev, A. (1911) Compt. Rend. 152, 49. 10. Jakoby, W. A. (1971) in Methods in Enzymology. Vol. XXII, p. 25, (Colowick, S. P., and Kaplan. N. 0.. eds.), Academic Press. New York. 11. Alberts, B. M., Amodio, M. J.. Gutman. E. I).. and Ferris, F. L. (1968) Cold Sprin,? Hurhor
The yeast photolyase is interesting from many points of view. It repairs uv-damaged genetically potent DNA and the nature of the lesion (substrate) is known to be primarily pyrimidine dimers (13- 1.5, 17). The enzyme binds to uv-exposed DNA in the dark (5). Exposure of the enzyme-uv-exposed DNA complex to the proper radiation (max at 370 nm) leads to a monomerization of the pyrimSymp. Quunr. Biol. 33, 289. idines in the lesion and a recovery of the 12. Harm, H.. and Rupert. C. S. (1968) Mttr. Re.s. specific genetic property (16). The cyclobutyl 6, 355-370. ring of the dimer is ruptured in this process. 13. Wulff, D. L., and Rupert. C. S. (1962) Biochenr. Biophys. Res. Commun. 7, 237-240. Since this involves the breaking of a carbon14. Setlow. J. K.. and Bollum. F. J. Biochim. Biophy.\. carbon bond with the aid of relatively low-enActu 157, 233-237. ergy radiation it suggests that the enzyme 15. Hayes, F. N., Williams, D. L.. Ratiff. R. L.. either places considerable strain on the subVarghere. A. J.. and Rupert. C. S. ( 1971) J. Amer. Chem. SW. 93, 4940. strate structure or that it binds selectively 16. Setlow, J. K. (196.5) in Current Topics in Radiation to a labile tautomeric form of the substrate. Research (Ebert. M., and Howard, A.. eds.). Extensive physicochemical studies of the p. 191, North Holland, Amsterdam. many properties of the enzyme by us await 17. Harm, W. (1978) Mur. Res. 51, 301-310. stabilization of the purified preparations. The 18. Bootwright, D. T.. Madden. J. J., Denson. J., and Werbin. H. (1975)Bio~hemisrrv. 14,5418-5421. heat treatment described here inflicted only
BIOCHEMISTRY
Purification
95, 77-81 (1979)
of the Photoreactivating
Enzyme from Yeast’
ROGER M. HERRIOTT Depurtment
of Biochemistry, School of Hygiene 615 North Wolje Street,
and Public Baltimorr.
Health. Murylund
The Johns 21205
Hopkins
University,
Received October 12. 1978 A method of purifying the DNA photolyase (EC 4.1.99.3) from yeast has been developed. The method includes one step which has been included to destroy enzymes that may be responsible for the instability of purified preparations.
unequivocal method. The general procedures for carrying out genetic transformation in this system have been described in detail elsewhere (6,7) but an improved method of making the wild-type cells competent is worth describing. Enzyme substrate. Purified transforming DNA carrying the resistance marker to 2000 pg/ml of streptomycin and capable of transforming 1 to 2 x 10” cells per microgram of DNA taken up by competent cells was diluted in saline-O.02 M PO1, pH 7.2, to a concentration of 2 &ml. Of this solution, 25 ml was placed in a 14-cm sterile petri dish at a distance of 55 cm from a 15-W GE sterilamp. Exposure time was 40 s, during which the dish was gently rotated. This exposure to approximately 1000 ergs/mm* reduced the transforming activity to 10 to 15% of value prior to exposure. This solution kept without change for several months at 5°C in a capped tube. The unit of enzyme activity (Eil~‘).~A unit of photoreactivating enzyme is defined as that quantity which raises the transforming activity of 1 j&ml of 10 to 15% active uvdamaged streptomycin-resistant H. influen?lae DNA at the rate of 1 x lo6 transformants per minute under defined conditions. The
Yeast as a source of the photoreactivating enzyme (DNA photolyase, EC 4.1.99.3) has the advantage over other sources in being readily available commercially in large relatively reproducible quantities. This advantage has been recognized by a number of investigators (l-4). Most of these workers found, however, that the stability of the enzyme decreased as it was purified. This writer, on finding that a variety of modifications in the medium produced no appreciable increase in stability, considered the possibility of a protease which accompanies the photolyase during purification and leads to the latter’s instability. A method of eliminating such a protease without a large loss of photolyase was developed based on Rupert’s finding (5) that the enzyme is protected against inactivation at 66°C by the presence of DNA exposed to ultraviolet light. Quantitative estimates of the stability of preparations from the new procedure have not been made. METHODS Enzyme assay. This assay consists of the initial rate of restoring the activity of uvinactivatedH. influenzae-transforming DNA carrying the genetic marker for high-level resistance to streptomycin. It is a long but
’ Abbreviations used: EU. enzyme unit, DTT. Dithiothreitol: HI, heart infusion; HI*, heart infusion plus 10 pg/ml of hemin and 2 to 4 &ml of DPN: TCA, trichloroacetic acid.
’ This paper is dedicated to the memory of Dr. Alvin Nason. 71
0003.2697/79/070077-05$02.00/O Copyright 6: 1979 by Academx Pre\\. Inc All r,ght\ of reproduct,on ,n itnv frxm re\rr\cd.
78
ROGERM.HERRIOlT
defined conditions include: a medium of 0.15 M NaCl-0.02 M phosphate, pH 7.5-2 mM dithiothreitol (DTT)-I mM EDTA and 5 x lop4 M reduced glutathione. One to two milliliters of enzyme and substrate were placed in a l-cm diameter test tube that was illuminated in a glass-bottom water bath kept at 37°C 1 to 2 cm from three parallel GE F20T 12 BL lamps. Diluent solutions. (a) For the enzyme: 100 &ml of bovine serum albumin-O.15 M saline-0.02 M P04, pH 7.5-2 mM DTT- 1 mM EDTA. (b) Diluent of DNA for assay: 0.15 M NaCl-0.02 M Na, citrate. Assay procedure. (Work in dim or yellow light.) (a) Dilute the enzyme in cold enzyme diluent to about 10 EU/ml and note the dilution factor. (b) Warm 2 ml of enzyme and illuminate at 37°C for 10 min; then chill and keep dark. This is to repair any uv lesions in the DNA that may be in the enzyme preparation. (c) To 1 ml of cold substrate, add 0.04 ml of 0.05 M reduced glutathione and 1 ml of enzyme in b; let stand 30 min cold and dark. (d) Warm tube c at 37°C for 5 min and remove a 0.1-ml sample, which will be diluted serially O.l/lO, then O.l/lO, in DNA diluent. This is the 0 time sample. (e) Turn on the illuminator and allow 5 min to reach a steady output. Place the test tubes with the enzyme-substrate mixture in the illuminated water bath and withdraw 0. I-ml samples after 2, 4, and 8 min of exposure and dilute as in d. (f) Put 0.2 ml of the last dilution of each sample into 1.6 ml of heart infusion, and follow with 0.2 ml of freshly thawed competent H. influenzae. Let stand 30 min at 37°C.
(g) Place 0.5 ml of this transformation mixture onto each of two petri dishes and add, with gentle mixing, 10 ml of warm (43°C) heart infusion*-agar (* means it has had hemin and DPN added to levels of 10 pg and 2 &ml, respectively). When the agar has solidified (15 min) the plates are incubated at 37°C for 1.5 h, and then overlayed with
another 10 ml of heart infusion*-agar containing 100 pg/ml of streptomycin sulfate. When the agar has set, incubate for 10 to 24 h and count the colonies. (In this assay only 50 pg/rnl streptomycin is used although the cells can grow in the presence of 2000 pg/ml.) (h) Control plates to be included: (1) no DNA but competent cells; (2) DNA but no competent cells. Neither control plates should show more than an occasional colony if the stocks are clean. (i) Sum the colony counts of the two plates for each sample and subtract any blanks or controls. Plot the difference in colony count against the time of illumination. The slope of the best straight line through the points times 1 x lo5 (the dilution factor) is now divided by 1 x lo6 to get the number of EU. (j) The number of EU/ml of initial solution is the number of EU found in i times 2x the dilution of enzyme in step 1. (The 2x is due to the dilution with substrate.) EXAMPLE: If the enzyme was diluted IOOOfold (103) and the plot showed a slope of 25 transformations per minute, then the EUlml of original is 25 x 1 x lo5
1 x 106
x 2 x IO3 or 5 x 103.
Preparation of competent (transformable) H. injluenzae. Although this process has
been described previously (6,7) some modifications or details deserve to be mentioned. Some of the stock solutions used in making M-IV solution are not as stable as appeared earlier. Thus, stock No. 22 keeps only a few weeks refrigerated. Stock No. 40 should be made of certified Difco casamino acids and it keeps only 10 days to 2 weeks. The dry casamino acids should be kept in a desiccator and the solution should be filter sterilized. The M-IV solution is best if kept only a week refrigerated. 1. Wild-type (streptomycin-sensitive) Rd H. injluenzae known to yield competent cultures were grown in heart infusion (HI) plus 10 pg/ml of hemin and 2 to 4 p.g/ml of DPN. The complete growth medium is designated
PURIFICATION
OF DNA
HI*. Stocks of such cells at a concentration of 1 x 10g/ml were frozen at -70°C after first adding glycerin to 15% or dimethyl sulfoxide to 20%. 2. A sample of frozen stock was diluted 102, 104. and lo6 in HI and 0.05 ml of each dilution was placed in 5 ml of HI* at 5 PM and rotated at 37°C overnight at about 200 rpm. Also included was a tube containing medium alone (HI*) and one of HI + cells but no hemin or DPN. 3. Early the next morning the two control tubes were clear, indicating that there was no contaminant in the system; 1 ml of the highest dilution showing a turbid culture was introduced into 50 ml of HI* in a 500-ml flask with a 28-mm side tube and rotated at 175 rpm in a New Brunswick water bath at 37°C. When the turbidity, compared to an HI* blank, reached 0.1 o.d. in a Coleman Jr. spectrophotometer set at 650 nm (usually about 2.5 h) the cells were centrifuged in a table model Sorvall SS- 1 centrifuge for 3 min at a powerstat setting of 27. In rapid succession the centrifuge was stopped manually, the supematant was decanted, and the cells were resuspended in 20 ml of warm M-IV; this suspension was centrifuged as before and stopped quickly; the supernatant was decanted away and cells were resuspended in 20 ml of warm M-IV and placed in a 250- to 300-ml Erlenmeyer flask and rotated for 100 min at 125 rpm at 37°C. The cells again were centrifuged at setting 27 and the cells were resuspended in 20 ml of cold HI* containing 15% glycerin or 20% dimethyl sulfoxide. Two to four milliliters of the cells were placed in sterile vials and frozen immediately at -70°C. The competence of cells prepared in this manner was usually greater than 1%. Most of the cells are competent but only 1% get the streptomycinresistance marker. Competence can be measured in several ways but for purposes of this paper the percentage of cells transformed to streptomycin resistance when an excess (1 &ml) of the srrep’ DNA was present (6) has been used. The frozen cells should be thawed quickly
79
PHOTOLYASE
just before use. The cells at -70°C hold their competence for up to 1 month. Microprotein determination. This procedure is for use when the concentration of protein is below 50 pg/ml. The reagents are those of Lowry et al. (8): (A). 10 ml of 2% Na,CO, + 0.1 ml of 1% CuSO, + 0.1 ml of 2.7% NaK tarn-ate; (B). 1 N Folin-Ciocalteu Reagent. To any volume of solution containing 5 to 25 ,ug of protein is added 0.25 vol of cold 50% TCA. Let stand for 10 min; then centrifuge for 10 min; decant carefully: wash 2x with 10% TCA unless you know there are no interferring contaminants such as -SH compounds. Dissolve the precipitate in 0.2 ml of 0.5 N NaOH. Add 1 ml of reagent A and let stand for 10 min; add 0.1 ml of reagent B with mixing and let stand at room temperature for 45 min. Read in l-ml cuvettes in a Beckman spectrophotometer at 700 nm. Run standards and a blank with each determination. RESULTS
The method of preparing the photolyase from yeast is described in Table 1 along with the analytical data indicating the recovery of enzyme activity. The protein content of fraction No. 5 was 6 to 10 mg/ml, so the specific activity (EU/mg of protein) was about 8000, or nearly 500-fold higher than that of a buffer extract of dried yeast. The average yield from several preparations was 42% of the initial activity. Solution No. 5 was stored frozen after the addition of 1 ml of glycerin-or carried on to the affinity chromatography step. Additional
Purijicution
Subsequent purification of the new preparations by affinity chromatography on an uv-exposed DNA cellulose columns ( 11) have not been repeated sufficiently to report the results at this time. Preparations that had no heat treatment but were precipitated by streptomycin sulfate in the presence of
3
4
5
The dialyzed material from No. 3 was mixed with 5 ml of 1.6 mg/ml calf thymus DNA in 0.1 M NaCl that had been exposed to 1 x lo6 ergs/mm* of uv radiation with stirring. (Twenty-five milliliters of DNA on 14-cm petri dish rotated 14 cm from GE Sterilamp for 40 min.) Let DNA + enzyme stand cold and dark for 1 h. Warm to 35°C in a liter beaker. Introduce a strong magnetic stirrer and add rapidly an equal volume of 0.05 M POd2-, pH 7.5-2 mM ME- 1 mM EDTA heated to 92°C. The temperature of the mixture rises to 60°C. Let stand 2 to 3 min; then chill in ice water. Centrifuge at 7K for 10 min. Save the supernatant.
Number 4 $ 2 vol of cold 1 mM ME-EDTA + 1 vol of 1.6% streptomycin sulfate; let stand 3 h or overnight at YC. Centrifuge for 10 min at 7K; discard supematant; dissolve residue in 10 ml of either 0.25 M PO,*-, pH 7.5 or 0.2 M KCI-0.05 M Tris-HCl-1 mM EDTA-5 mM DTT-0.02 M PO,*--5% glycerine, pH 7.5. This latter solvent is used when further purification by affinity chromatography on a DNA column exposed to uv is to follow.
g) was stirred in to bring the AS concentration to discard the supematant and dissolve the residue in mM EDTA. Dialyze this overnight in washed (10) same solvent in a tall jar with stirring. Open the
Supematant No. 0.55 saturated. 206 ml of cold s-in. Visking dialysis bags.
2 + 130 g/liter of solid AS (or 400 Centrifuged at 7K for 20 to 30 mitt; 0.05 M P0,2m, pH 7.5-2 mM ME-l tubing against 6 to 7 liters of the
2
Dried yeast (700 g) mixed slowly with 3.5 liters of warm 0.1 M K,HPO,-5 m&t mercaptoethanol (ME)- 1 mM EDTA in a 6-liter flask. Toluene (22 ml) was added and stirred with a magnetic stirrer at 37°C for 4 h. The solution was chilled by placing the flask in cold water; 735 g of solid ammonium sulfate (AS) was added and stirred slowly until the AS had dissolved. After standing overnight the precipitate was centrifuged at 7K for 0.5 h in a Sorvall 2B centrifuge. The supematant was saved.
No.
1
OFPHOTOLYASE
1
Fresh baker’s yeast was air-dried by crumbling it to small particles on brown wrapping paper and allowing it to stand on the laboratory desk. It was stored at 32°F or lower. This is the procedure of von Lebedev (9) and was used by Rupert (1).
Procedures and materials
PARTIALPURIFICATION
TABLE
10
750
400
2900
Volume
50,000
650
2000
400
Enzyme units per milliliter
5 x 105
5 x 105
Total enzyme units
PURIFICATION
uv-exposed DNA have been chromatographed on such columns after first separating the enzyme from the uv-exposed DNA by ammonium sulfate precipitation. This yielded another 50-fold rise in specific activity. When this was followed by passage through a column of unirradiated DNA more protein was removed with a 2- to 3-fold increase in specific activity. These preparations were unstable perhaps because they were so dilute (3 pg of protein/ml) or because of the hypothetical protease which the present method should reduce. The preparations of highest specific activity (IO5 above an aqueous extract of the yeast) were similar in many ways to those reported by Werbin’s group (4, 18). This purification is encouragingly close to a figure calculated by Harm and Rupert from their photoflash studies (12). There was an absorption maximum at 370 nm in the preparations of high specific activity as well as one at 280 nm. There was also a strong fluorescence emission maximum at 470 nm when exposed to 360-nm radiation. DISCUSSION
81
OF DNA PHOTOLYASE
a small loss in photolyase activity, as expected from Rupert’s earlier studies (5). It remains to be determined if the hoped for stabilization of highly purified preparation has been realized. ACKNOWLEDGMENT The writer acknowledges with gratitude the assistance received in this and related studies by postdoctoral associates Sang Won Park and William Hauswirth, by Margarita Jiminez, and by technical assistants Genny Armilei, Lynne Vique, Robert Union, and Sandra Richter Takai. The competence procedure was examined by Matthew Buechner. This investigation was supported by Public Health Service Research Grant AI-01218 from the NIAID and by Atomic Energy Commission Contract AT(30- 1) I37 I (NYD-1371-58).
REFERENCES 1. Rupert, C. S. (1960) J. Gen. Physiol. 43, 573. 2. Muhammed, A. (1966) J. Biol. Chrm. 241, 516523. 3. Setlow, J. K., Personal communication. 4. Minato, S.. and Werbin, H. (1971) B&hemi.rtrj 10, 4503-4508. 5. Rupert. C. S. (1962)J. Gen. Physiol. 45,725-741. 6. Herriott, R. M., Meyer, E. Y., and Vogt, M. (1970) J. Bacterial. 101, 517-524. 7. Herriott, R. M. (1971) in Chemical Mutagens (Hollaender. A., ed.). pp. 175-212. Plenum Press. New York. 8. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall. R. J. (1951) J. Biol. Chem. 193, 265. 9. von Lebedev, A. (1911) Compt. Rend. 152, 49. 10. Jakoby, W. A. (1971) in Methods in Enzymology. Vol. XXII, p. 25, (Colowick, S. P., and Kaplan. N. 0.. eds.), Academic Press. New York. 11. Alberts, B. M., Amodio, M. J.. Gutman. E. I).. and Ferris, F. L. (1968) Cold Sprin,? Hurhor
The yeast photolyase is interesting from many points of view. It repairs uv-damaged genetically potent DNA and the nature of the lesion (substrate) is known to be primarily pyrimidine dimers (13- 1.5, 17). The enzyme binds to uv-exposed DNA in the dark (5). Exposure of the enzyme-uv-exposed DNA complex to the proper radiation (max at 370 nm) leads to a monomerization of the pyrimSymp. Quunr. Biol. 33, 289. idines in the lesion and a recovery of the 12. Harm, H.. and Rupert. C. S. (1968) Mttr. Re.s. specific genetic property (16). The cyclobutyl 6, 355-370. ring of the dimer is ruptured in this process. 13. Wulff, D. L., and Rupert. C. S. (1962) Biochenr. Biophys. Res. Commun. 7, 237-240. Since this involves the breaking of a carbon14. Setlow. J. K.. and Bollum. F. J. Biochim. Biophy.\. carbon bond with the aid of relatively low-enActu 157, 233-237. ergy radiation it suggests that the enzyme 15. Hayes, F. N., Williams, D. L.. Ratiff. R. L.. either places considerable strain on the subVarghere. A. J.. and Rupert. C. S. ( 1971) J. Amer. Chem. SW. 93, 4940. strate structure or that it binds selectively 16. Setlow, J. K. (196.5) in Current Topics in Radiation to a labile tautomeric form of the substrate. Research (Ebert. M., and Howard, A.. eds.). Extensive physicochemical studies of the p. 191, North Holland, Amsterdam. many properties of the enzyme by us await 17. Harm, W. (1978) Mur. Res. 51, 301-310. stabilization of the purified preparations. The 18. Bootwright, D. T.. Madden. J. J., Denson. J., and Werbin. H. (1975)Bio~hemisrrv. 14,5418-5421. heat treatment described here inflicted only
