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[133a] DNA methylase from Escherichia coli
[133a]
DNA. METHYLASE
491
example, cell e x t r a c t prepared from E. coli do not catalyze detectable methylation of E. coli sRNA but transfer methyl groups to sRNA isolated from Micrococcus lysodeikticus, Clostridium pasteurianum, etc. Cell extracts prepared from M. lysodeikticus catalyze methylation of sRNA from E. coli and C. pasteurianum but are not active when measured with sRNA from M. lysodeikticus. These observations are in keeping with the idea that there are a limited number of sites available for methylation and that these sites (presumably nucleotide sequences) are species and strain specific. Properties of the Purified Enzymes
Effect o] pH on the Rate o] Reaction. Optimal activities for the different methylating enzymes were found in the following pH ranges: guanine I, 8.0-8.5 in both Tris and triethanolamine buffers; guanine II, and cytosine, triethanolamine buffer, pH 8.0-9.0; adenine and uracil, triethanolamine buffer, pH 8.5-9.0; and guanine III, dimethylglutarate buffer, pH 7.5-8.0. Requirements for Metals and Sulfhydryl Agents. All enzymes showed some activity in the absence of Mg++; the methylation of cytosine was not affected by Mg ÷+, while the uracil-methylating and the guaninemethylating activities I and II were increased approximately 4-fold, and guanine-methylating enzyme III, 2-fold, by the presence of Mg ~. Methylation of adenine was stimulated only 2-fold by Mg ÷÷. The enzyme preparations were routinely stored in the presence of 2-mercaptoethanol; in some cases, there was a marked decrease in methylation activity when this compound was omitted from the reaction mixtures, especially in the case of the uracil-methylating enzyme. The addition of p-hydroxymercuribenzoate decreased the activity of all the enzyme fractions tested.
[ 1 3 3 a ] D N A M e t h y l a s e f r o m Escherichia coli 1
By MARVIN GOLD and JERARDHURWITZ DNA W S-adenosylmethionine(14C-methyl-labeled)--* I~C-HpDNA -b S-adenosylhomocysteine Assay Method
Principle. The assay is based on the incorporation of radioactivity into an acid-insoluble product. 1M. Gold and J. Hurwitz, J. Biol. Chem. 239, 3858 (1964).
492
MODIFICATION OF NUCLEIC ACIDS
[133a]
Reagents. The reaction mixture (total volume 0.25 ml) contains: Tris buffer, 0.1 M, pH 8.0 2-Mercaptoethanol, 0.016 M 14C-CHs-labeled S-adenosylmethionine, 0.04 mM, (2 to 4 X 107 cpm per mieromole) DNA, 0.4 m M (in terms of nueleotide residues). For routine assays, DNA isolated from either Micrococcus lysodeikticus or Mycobacterium phlei may be used Enzyme The other reagents used are: Perehlorie acid, 7 ~ Bovine plasma albumin, 0.5% Sodium pyrophosphate, 0.1 M Perchlorie acid, 2%, containing 0.002 M sodium pyrophosphate NH4OH, 2 M
Procedure. After 30 minutes of incubation at 37 °, the reaction is stopped and the DNA is precipitated with 0.4 ml of ice-cold perchloric acid. Bovine plasma albumin (0.05 ml) is added as a carrier to facilitate complete precipitation; and sodium pyrophosphate (0.1 ml), to prevent the nonspecifie binding of unreacted S-adenesylmethionine. After 2 minutes at 0 ° with occasional shaking, the suspensions are centrifuged for 1 minute at 15,000 g; the supernatants are discarded. The pellets are then finely suspended in the 2% perchloric acid with a tight-fitting glass pestle, and the suspensions are centrifuged as above. This procedure is repeated once more, and the pellets are dissolved with 1.5 ml of ammonium hydroxide. The solutions are decanted into metal planehets, dried under an infrared lamp, and counted in a windowless gas-flow counter. A blank without enzyme or DNA is included, and its value is subtracted from each sample; these blank values are routinely less than 1 mieromicromole of 14C-methyl group incorporated. One unit of DNA methylase activity is defined as that amount catalyzing the incorporation of 1 millimicromole of methyl group in 30 minutes. Specific activity is defined as units per milligram of protein. Under the conditions described, and using M. lysodeikticus DNA, the rate of methylation is proportional to enzyme concentration up to 1 unit. The rate is also proportional to time of incubation until a fixed yield is attained (see below).
[133a]
DNA METHYI~SE
493
Isolation Procedure
Growth o] Cells. Escherichia coli strain W was grown and harvested as previously described} a All subsequent operations are carried out at 0-5 °, and all solutions should contain 0.005M 2-mercaptoethanol and 0.001 M EDTA. Crude Extract. Frozen E. coli, strain W (200 g), was ground with 400 g of Alumina A-301 (Alcoa) in a preehilled mortar kept in an ice bath. When the loose consistency of the mixture indicated that cell breakage was complete, it was stirred with 400 ml of 0.05 M triethanolamine buffer, pH 8.8, containing 0.01 M MgCl~. The suspension was centrifuged for 20 minutes at 12,000 g, and the pellet was reextracted with 300 ml of the same buffer and centrifuged. The two supernatant solutions were combined and centrifuged for 2 hours at 30,000 rpm in the No. 30 rotor of the Spinco preparative ultracentrifuge. The supernatant solution (crude extract, 660 ml) was stored in the refrigerator overnight. Alumina C~ Gel Eluate. To 650 ml of the crude extract were added 260 ml of alumina C~ gel (17.9 mg of solids per milliliter), and the suspension was mixed. After 20 minutes, the gel was removed by centrifugation at 5000 g for 10 minutes; the supernatant solution was discarded. The gel was washed by homogenization with 325 ml of 0.05 M potassium phosphate buffer, pH 7.5. After 10 minutes, the suspension was centrifuged at 5000 g for 10 minutes; the supernatant fluid was discarded. This procedure was repeated twice more. The DNA-methylating activity was then eluted from the gel by homogenization in 225 ml of 0.5 M potassium phosphate buffer, pit 7.5, and centrifuged at 5000 g for 10 minutes. The pellet was reextracted with 0.5 M potassium phosphate buffer, and the two supernatant solutions were combined to give the alumina C~ gel eluate (450 ml). Calcium Phosphate Gel Eluate. The alumina C~ gel eluate was diluted with water to a volume of 4500 ml, and 450 ml of calcium phosphate gel (12.1 mg of solids per milliliter) was added with stirring. After 30 minutes, the suspension was centrifuged and the supernatant fluid was discarded. The gel was washed by homogenization with 1 liter of 0.1 M potassium phosphate buffer, pH 6.5, and centrifuged. The supernatant solution was discarded and the gel was washed once more with 500 ml of the same buffer. The activity was then eluted from the gel with two homogenizations with 100-ml portions of 0.5 M potassium "J. J. Furth, J. tturwitz, R. Krug, and M. Alexander, J. Biol. Chem. 236, 3317 (1961).
494
MODIFICATION OF NUCLEIC ACIDS
[133a]
fl, fl-dimethylglutarate buffer, pH 7.5. These two washings were combined (calcium phosphate gel eluate). Ammonium Sulfate I. The combined eluates were immediately further fractionated with ammonium sulfate. To the calcium phosphate gel eluate (194 ml), 104 ml of saturated ammonium sulfate solution (prepared at room temperature and having a pH of 7.4 after 100-fold dilution) was added. After 30 minutes, the suspension was centrifuged for 15 minutes at 30,000 rpm in the No. 30 rotor of the Spinco Model L ultracentrifuge, and the pellet was discarded. The supernatant fluid was brought to 65% saturation by the addition of 55.2 g of solid ammonium sulfate. After 30 minu~es, the suspension was centrifuged and the supernatant solution was discarded. The pellet was dissolved in 0.02 M potassium phosphate buffer, pH 7.5, to give ammonium sulfate fraction I (volume, 10.6 ml). Phosphocellulose Chromatography. Phosphocellulose was suspended in water and the fines were removed. The phosphocellulose was then poured as a slurry into a column 2.5 cm in diameter and packed by pressure to a height of 20 cm. The column was washed with 0.02 M potassium phosphate buffer, pH 7.5, until the pH of the effluent was 7.5. Ammonium sulfate fraction I was dialyzed against 1 liter of the same buffer for 40 minutes; the dialysis fluid was then replaced with 1 liter of fresh buffer, and the dialysis was continued for a further 45 minutes. The volume of ammonium sulfate fraction I increased to 13 ml, and the slight precipitate which formed inside the dialysis bag was not removed. The dialyzed fraction was applied to the top of the column, which was then washed successively with the following potassium phosphate buffers, pH 7.5:50 ml of 0.02 M, 100 ml of 0.1M, 150 ml of 0.2M, and 150 ml of 0.5 M. The flow rate was approximately 5 ml per minute, and 25-ml fractions were collected. Most of the enzymatic activity eluted from the column appeared in the third 0.5M fraction, and this was retained for further purification (phosphocellulose eluate, 25 ml). Ammonium Sul]ate II. Solid ammonium sulfate (9 g) was added to the phosphocellulose fraction (25 ml), and after 15 minutes, the suspension was centrifuged for 10 minutes at 40,000 rpm in the No. 40 rotor of the Spinco Model L ultracentrifuge. The pellet was then successively extracted by homogenization in ammonium sulfate solutions of various concentrations containing 0.05 M potassium phosphate buffer, pH 7.5, as follows: 10 ml of 6 0 ~ saturated, 10 ml of 50%, and 1.5 ml of 309. After each extraction, the suspension was allowed to stand for 10 minutes before it was centrifuged. Over 959 of the enzymatic activity was found in the last fraction (ammonium sulfate II, 1.5 ml).
[133a]
DNA METHYLASE
495
PURIFICATION OF DNA METHYLASEACTIVITYa
Enzyme fraction
Total units
Protein (mg/ml)
Specific activity (units/mg protein)
Crude extract Alumina C~ gel eluate Calcium phosphate gel eluate Ammoniumsulfate I Phosphocellulose eluate Ammonium.sulfateII
1980 1710 1240 1000 655 300
13.4 2.8 0.53 4.5 0.44 2.3
0.22 1.36 12.0 22.2 59 87
Assayed with M. lysodeikticus DNA. A summary of the purification is given in the table. The activity has been purified approximately 400-fold with a yield of 15%. Properties of the Enzyme Purity. The final preparation still contains small amounts of the sRNA methylases.2 Deoxyribonuelease activity can be detected only by the destruction of biologically active DNA. Requirements. The reaction does not require the presence of divalent metal ions and, in fact, can be assayed in the presence of relatively high concentrations (0.02M) of EDTA. In crude extracts, the presence of EDTA enhances the activity. If 2-mercaptoethanol is omitted from the reaction mixture, the activity of the enzyme is decreased by 90%. The requirement for S-adenosylmethionine cannot be met by other methyl donors, 1 and S-adenosylethionine is inactive. S-Adenosylhomocysteine is a potent inhibitor of the reaction. Most DNA preparations from viral, bacterial, plant, or animal sources can serve as methyl group acceptors2 -5 Mononucleotides, RNA or synthetic polydeoxynucleotides of known base sequence are inactive. Products. In most DNA preparations, the sites of methylation are cytosine and adenine, yielding moieties of 5-methylcytosine and 6-methylaminopurine, respectively. The products are identified routinely by standard methods of base analysis. Nature of Reaction. With any given DNA, the reaction proceeds zj. Hurwitz, M. Gold, and M. Anders, J. Biol. Chem. 239, 3462 (1964). M. Gold, J. Hurwitz, and M. Anders, Biochem. Biophys. Res. Commun. 11, 107 (1963). M. Gold and J. Hurwitz, Cold Spring Harbor Syrup. Quant. Biol. 27, 149 (1963). M. Gold, J. Hurwitz, and M. Anders, PToc. Natl. Aead. Sei. U.S. 50, 164 (1963).
496
MODIFICATION OF NUCLEIC ACIDS
[133b]
until a finite number of methyl groups have been incorporated. The addition of further excess enzyme at this point is without effect, and the reactions appear to be irreversible. The yield of methylation appears to be a characteristic of the DNA and can reach levels as high as 2--5% of the total nueleotide residues with such DNA's as Mycobacterium phlei, Micrococcus lysodeikticus, and Pseudomonas aeruginosa. The relative amount of 5-methylcytosine and 6-methylaminopurine formed is also a characteristic of any given DNA. Both of the above properties appear to be independent of base composition. Although DNA partially degraded by nucleases or sonic oscillation can serve as an acceptor, heat-denatured DNA is inactive B DNA isolated from the same strain as the enzyme is also inactive2,4 Other Sources. The enzyme may be purified from other strains of E. coli by the procedure given above. It should be noted that K12 strains contain activity for both cytosine and adenine while B strains can form only 6-methylaminopurine. After infection with certain bacteriophage, the activity of DNA methylase in cell extracts increases markedly 7 and can be purified by a slight modification of the above procedure. Stability. Except for the calcium phosphate gel eluate, all enzyme fractions can be stored at 0-5 ° for several days without significant loss of activity. The ammonium sulfate fractions are unstable during prolonged dialysis. 6M. Gold and J. Hurwitz, J. Biol. Chem. 239, 3866 (1964). 'M. Gold, R. Hausmann, U. Maitra, and J. Hurwitz, Proc. Natl. Acad. 8cl. U.S. 52, 292 (1964).
[133b]
I s o l a t i o n a n d P r o p e r t i e s o f E n z y m e s I n v o l v e d in Glycosylation of Bacteriophage DNA
By JOHN JOSSE Introduction The enzymes to be described have all been isolated from Escherichia coli cells infected with bacteriophages T2, T4, or T6. As summarized in Table I, adapted from the work of Lehman and colleagues, the DNA's of these phages contain characteristic amounts of glucose, covalently bound to 5-hydroxymethylcytosine (HMC) residues. 1 The distribution 1Abbreviations used are : HMC, 5-hydroxymethylcytosine; G, D-glucose; ~4C-UDPglucose, 14C-glucose-labeledUDP-glucose.
DNA. METHYLASE
491
example, cell e x t r a c t prepared from E. coli do not catalyze detectable methylation of E. coli sRNA but transfer methyl groups to sRNA isolated from Micrococcus lysodeikticus, Clostridium pasteurianum, etc. Cell extracts prepared from M. lysodeikticus catalyze methylation of sRNA from E. coli and C. pasteurianum but are not active when measured with sRNA from M. lysodeikticus. These observations are in keeping with the idea that there are a limited number of sites available for methylation and that these sites (presumably nucleotide sequences) are species and strain specific. Properties of the Purified Enzymes
Effect o] pH on the Rate o] Reaction. Optimal activities for the different methylating enzymes were found in the following pH ranges: guanine I, 8.0-8.5 in both Tris and triethanolamine buffers; guanine II, and cytosine, triethanolamine buffer, pH 8.0-9.0; adenine and uracil, triethanolamine buffer, pH 8.5-9.0; and guanine III, dimethylglutarate buffer, pH 7.5-8.0. Requirements for Metals and Sulfhydryl Agents. All enzymes showed some activity in the absence of Mg++; the methylation of cytosine was not affected by Mg ÷+, while the uracil-methylating and the guaninemethylating activities I and II were increased approximately 4-fold, and guanine-methylating enzyme III, 2-fold, by the presence of Mg ~. Methylation of adenine was stimulated only 2-fold by Mg ÷÷. The enzyme preparations were routinely stored in the presence of 2-mercaptoethanol; in some cases, there was a marked decrease in methylation activity when this compound was omitted from the reaction mixtures, especially in the case of the uracil-methylating enzyme. The addition of p-hydroxymercuribenzoate decreased the activity of all the enzyme fractions tested.
[ 1 3 3 a ] D N A M e t h y l a s e f r o m Escherichia coli 1
By MARVIN GOLD and JERARDHURWITZ DNA W S-adenosylmethionine(14C-methyl-labeled)--* I~C-HpDNA -b S-adenosylhomocysteine Assay Method
Principle. The assay is based on the incorporation of radioactivity into an acid-insoluble product. 1M. Gold and J. Hurwitz, J. Biol. Chem. 239, 3858 (1964).
492
MODIFICATION OF NUCLEIC ACIDS
[133a]
Reagents. The reaction mixture (total volume 0.25 ml) contains: Tris buffer, 0.1 M, pH 8.0 2-Mercaptoethanol, 0.016 M 14C-CHs-labeled S-adenosylmethionine, 0.04 mM, (2 to 4 X 107 cpm per mieromole) DNA, 0.4 m M (in terms of nueleotide residues). For routine assays, DNA isolated from either Micrococcus lysodeikticus or Mycobacterium phlei may be used Enzyme The other reagents used are: Perehlorie acid, 7 ~ Bovine plasma albumin, 0.5% Sodium pyrophosphate, 0.1 M Perchlorie acid, 2%, containing 0.002 M sodium pyrophosphate NH4OH, 2 M
Procedure. After 30 minutes of incubation at 37 °, the reaction is stopped and the DNA is precipitated with 0.4 ml of ice-cold perchloric acid. Bovine plasma albumin (0.05 ml) is added as a carrier to facilitate complete precipitation; and sodium pyrophosphate (0.1 ml), to prevent the nonspecifie binding of unreacted S-adenesylmethionine. After 2 minutes at 0 ° with occasional shaking, the suspensions are centrifuged for 1 minute at 15,000 g; the supernatants are discarded. The pellets are then finely suspended in the 2% perchloric acid with a tight-fitting glass pestle, and the suspensions are centrifuged as above. This procedure is repeated once more, and the pellets are dissolved with 1.5 ml of ammonium hydroxide. The solutions are decanted into metal planehets, dried under an infrared lamp, and counted in a windowless gas-flow counter. A blank without enzyme or DNA is included, and its value is subtracted from each sample; these blank values are routinely less than 1 mieromicromole of 14C-methyl group incorporated. One unit of DNA methylase activity is defined as that amount catalyzing the incorporation of 1 millimicromole of methyl group in 30 minutes. Specific activity is defined as units per milligram of protein. Under the conditions described, and using M. lysodeikticus DNA, the rate of methylation is proportional to enzyme concentration up to 1 unit. The rate is also proportional to time of incubation until a fixed yield is attained (see below).
[133a]
DNA METHYI~SE
493
Isolation Procedure
Growth o] Cells. Escherichia coli strain W was grown and harvested as previously described} a All subsequent operations are carried out at 0-5 °, and all solutions should contain 0.005M 2-mercaptoethanol and 0.001 M EDTA. Crude Extract. Frozen E. coli, strain W (200 g), was ground with 400 g of Alumina A-301 (Alcoa) in a preehilled mortar kept in an ice bath. When the loose consistency of the mixture indicated that cell breakage was complete, it was stirred with 400 ml of 0.05 M triethanolamine buffer, pH 8.8, containing 0.01 M MgCl~. The suspension was centrifuged for 20 minutes at 12,000 g, and the pellet was reextracted with 300 ml of the same buffer and centrifuged. The two supernatant solutions were combined and centrifuged for 2 hours at 30,000 rpm in the No. 30 rotor of the Spinco preparative ultracentrifuge. The supernatant solution (crude extract, 660 ml) was stored in the refrigerator overnight. Alumina C~ Gel Eluate. To 650 ml of the crude extract were added 260 ml of alumina C~ gel (17.9 mg of solids per milliliter), and the suspension was mixed. After 20 minutes, the gel was removed by centrifugation at 5000 g for 10 minutes; the supernatant solution was discarded. The gel was washed by homogenization with 325 ml of 0.05 M potassium phosphate buffer, pH 7.5. After 10 minutes, the suspension was centrifuged at 5000 g for 10 minutes; the supernatant fluid was discarded. This procedure was repeated twice more. The DNA-methylating activity was then eluted from the gel by homogenization in 225 ml of 0.5 M potassium phosphate buffer, pit 7.5, and centrifuged at 5000 g for 10 minutes. The pellet was reextracted with 0.5 M potassium phosphate buffer, and the two supernatant solutions were combined to give the alumina C~ gel eluate (450 ml). Calcium Phosphate Gel Eluate. The alumina C~ gel eluate was diluted with water to a volume of 4500 ml, and 450 ml of calcium phosphate gel (12.1 mg of solids per milliliter) was added with stirring. After 30 minutes, the suspension was centrifuged and the supernatant fluid was discarded. The gel was washed by homogenization with 1 liter of 0.1 M potassium phosphate buffer, pH 6.5, and centrifuged. The supernatant solution was discarded and the gel was washed once more with 500 ml of the same buffer. The activity was then eluted from the gel with two homogenizations with 100-ml portions of 0.5 M potassium "J. J. Furth, J. tturwitz, R. Krug, and M. Alexander, J. Biol. Chem. 236, 3317 (1961).
494
MODIFICATION OF NUCLEIC ACIDS
[133a]
fl, fl-dimethylglutarate buffer, pH 7.5. These two washings were combined (calcium phosphate gel eluate). Ammonium Sulfate I. The combined eluates were immediately further fractionated with ammonium sulfate. To the calcium phosphate gel eluate (194 ml), 104 ml of saturated ammonium sulfate solution (prepared at room temperature and having a pH of 7.4 after 100-fold dilution) was added. After 30 minutes, the suspension was centrifuged for 15 minutes at 30,000 rpm in the No. 30 rotor of the Spinco Model L ultracentrifuge, and the pellet was discarded. The supernatant fluid was brought to 65% saturation by the addition of 55.2 g of solid ammonium sulfate. After 30 minu~es, the suspension was centrifuged and the supernatant solution was discarded. The pellet was dissolved in 0.02 M potassium phosphate buffer, pH 7.5, to give ammonium sulfate fraction I (volume, 10.6 ml). Phosphocellulose Chromatography. Phosphocellulose was suspended in water and the fines were removed. The phosphocellulose was then poured as a slurry into a column 2.5 cm in diameter and packed by pressure to a height of 20 cm. The column was washed with 0.02 M potassium phosphate buffer, pH 7.5, until the pH of the effluent was 7.5. Ammonium sulfate fraction I was dialyzed against 1 liter of the same buffer for 40 minutes; the dialysis fluid was then replaced with 1 liter of fresh buffer, and the dialysis was continued for a further 45 minutes. The volume of ammonium sulfate fraction I increased to 13 ml, and the slight precipitate which formed inside the dialysis bag was not removed. The dialyzed fraction was applied to the top of the column, which was then washed successively with the following potassium phosphate buffers, pH 7.5:50 ml of 0.02 M, 100 ml of 0.1M, 150 ml of 0.2M, and 150 ml of 0.5 M. The flow rate was approximately 5 ml per minute, and 25-ml fractions were collected. Most of the enzymatic activity eluted from the column appeared in the third 0.5M fraction, and this was retained for further purification (phosphocellulose eluate, 25 ml). Ammonium Sul]ate II. Solid ammonium sulfate (9 g) was added to the phosphocellulose fraction (25 ml), and after 15 minutes, the suspension was centrifuged for 10 minutes at 40,000 rpm in the No. 40 rotor of the Spinco Model L ultracentrifuge. The pellet was then successively extracted by homogenization in ammonium sulfate solutions of various concentrations containing 0.05 M potassium phosphate buffer, pH 7.5, as follows: 10 ml of 6 0 ~ saturated, 10 ml of 50%, and 1.5 ml of 309. After each extraction, the suspension was allowed to stand for 10 minutes before it was centrifuged. Over 959 of the enzymatic activity was found in the last fraction (ammonium sulfate II, 1.5 ml).
[133a]
DNA METHYLASE
495
PURIFICATION OF DNA METHYLASEACTIVITYa
Enzyme fraction
Total units
Protein (mg/ml)
Specific activity (units/mg protein)
Crude extract Alumina C~ gel eluate Calcium phosphate gel eluate Ammoniumsulfate I Phosphocellulose eluate Ammonium.sulfateII
1980 1710 1240 1000 655 300
13.4 2.8 0.53 4.5 0.44 2.3
0.22 1.36 12.0 22.2 59 87
Assayed with M. lysodeikticus DNA. A summary of the purification is given in the table. The activity has been purified approximately 400-fold with a yield of 15%. Properties of the Enzyme Purity. The final preparation still contains small amounts of the sRNA methylases.2 Deoxyribonuelease activity can be detected only by the destruction of biologically active DNA. Requirements. The reaction does not require the presence of divalent metal ions and, in fact, can be assayed in the presence of relatively high concentrations (0.02M) of EDTA. In crude extracts, the presence of EDTA enhances the activity. If 2-mercaptoethanol is omitted from the reaction mixture, the activity of the enzyme is decreased by 90%. The requirement for S-adenosylmethionine cannot be met by other methyl donors, 1 and S-adenosylethionine is inactive. S-Adenosylhomocysteine is a potent inhibitor of the reaction. Most DNA preparations from viral, bacterial, plant, or animal sources can serve as methyl group acceptors2 -5 Mononucleotides, RNA or synthetic polydeoxynucleotides of known base sequence are inactive. Products. In most DNA preparations, the sites of methylation are cytosine and adenine, yielding moieties of 5-methylcytosine and 6-methylaminopurine, respectively. The products are identified routinely by standard methods of base analysis. Nature of Reaction. With any given DNA, the reaction proceeds zj. Hurwitz, M. Gold, and M. Anders, J. Biol. Chem. 239, 3462 (1964). M. Gold, J. Hurwitz, and M. Anders, Biochem. Biophys. Res. Commun. 11, 107 (1963). M. Gold and J. Hurwitz, Cold Spring Harbor Syrup. Quant. Biol. 27, 149 (1963). M. Gold, J. Hurwitz, and M. Anders, PToc. Natl. Aead. Sei. U.S. 50, 164 (1963).
496
MODIFICATION OF NUCLEIC ACIDS
[133b]
until a finite number of methyl groups have been incorporated. The addition of further excess enzyme at this point is without effect, and the reactions appear to be irreversible. The yield of methylation appears to be a characteristic of the DNA and can reach levels as high as 2--5% of the total nueleotide residues with such DNA's as Mycobacterium phlei, Micrococcus lysodeikticus, and Pseudomonas aeruginosa. The relative amount of 5-methylcytosine and 6-methylaminopurine formed is also a characteristic of any given DNA. Both of the above properties appear to be independent of base composition. Although DNA partially degraded by nucleases or sonic oscillation can serve as an acceptor, heat-denatured DNA is inactive B DNA isolated from the same strain as the enzyme is also inactive2,4 Other Sources. The enzyme may be purified from other strains of E. coli by the procedure given above. It should be noted that K12 strains contain activity for both cytosine and adenine while B strains can form only 6-methylaminopurine. After infection with certain bacteriophage, the activity of DNA methylase in cell extracts increases markedly 7 and can be purified by a slight modification of the above procedure. Stability. Except for the calcium phosphate gel eluate, all enzyme fractions can be stored at 0-5 ° for several days without significant loss of activity. The ammonium sulfate fractions are unstable during prolonged dialysis. 6M. Gold and J. Hurwitz, J. Biol. Chem. 239, 3866 (1964). 'M. Gold, R. Hausmann, U. Maitra, and J. Hurwitz, Proc. Natl. Acad. 8cl. U.S. 52, 292 (1964).
[133b]
I s o l a t i o n a n d P r o p e r t i e s o f E n z y m e s I n v o l v e d in Glycosylation of Bacteriophage DNA
By JOHN JOSSE Introduction The enzymes to be described have all been isolated from Escherichia coli cells infected with bacteriophages T2, T4, or T6. As summarized in Table I, adapted from the work of Lehman and colleagues, the DNA's of these phages contain characteristic amounts of glucose, covalently bound to 5-hydroxymethylcytosine (HMC) residues. 1 The distribution 1Abbreviations used are : HMC, 5-hydroxymethylcytosine; G, D-glucose; ~4C-UDPglucose, 14C-glucose-labeledUDP-glucose.