JouRNAL F BACrjEuOLOGY, Apr. 1975, p. 129-138 Copyright @ 1975 American Society for Microbiology
Vol. 122, No. 1 Printed in U.S.A.
Deoxyribonucleic Acid-Cytosine Methylation by Host- and Plasmid-Controlled Enzymes MAUREEN S. MAY AND STANLEY HAFTMAN*
University of Rochester, Department of Biology, Rochester, New York
14627
Received for publication 17 January 1975
Deoxyribonucleic acid (DNA)-cytosine methylation specified by the wild-type Escherichia coli K12 mec+ gene and by the N-3 drug resistance (R) factor was studied in vivo and in vitro. Phage A and fd were propagated in the presence of L- [methyl-'H]methionine in various host bacteria. The in vivo labeled DNA was isolated from purified phage and depurinated by formic acid-diphenylamine treatment. The resulting pyrimidine oligonucleotide tracts were separated according to size and base composition by chromatography on diethylaminoethyl-cellulose in 7 M urea at pH 5.5 and 3.5, respectively. The distribution of labeled 5-methylcytosine in DNA pyrimidine tracts was identical for phage grown in mec+ and mec- (N-3) cells. For phage A the major 5-methylcytosinecontaining tract was the tripyrimidine, C2T; for both fd- mec- (N-3) DNA and fd mec+ DNA, C2T was the sole 5-methylcytosine-containing tract. When various A DNAs were methylated to saturation in vitro by crude extracts from mec+ and mec- (N-3) cells, the extent of cytosine methylation was the same. This is in contrast to in vivo methylation where A mec (N-3) DNA contains twice as many 5-methylcytosines per genome as A mec+ DNA. Therefore, we suggest that the K12 met+ cytosine methylase and the N-3 plasmid modification methylase are capable of recognizing the same nucleotide sequences, but that the in vivo methylation rate is lower in mec+ cells.
Many bacterial strains and some plasmids control deoxyribonucleic acid (DNA) restriction modification systems (2, 7). These systems enable a strain to protect its own DNA while foreign DNA is degraded. Modification is brought about by an adenine- or cytosinemethylase which methylates specific sequences on double-stranded DNA. Restriction occurs via an endonuclease which cleaves unmodified double-stranded DNA. The N-3 plasmid, a fi- drug resistance factor related to the R-15 plasmid, controls hspH host specificity (3, 4, 27, 29). This host specificity system is based on DNA-cytosine methylation (16, 25). The N-3 plasmid can restrict and/or modify double-stranded DNA phages, including coliphage X. The singlestranded DNA phages, fd and M13, are not subject to N-3 restriction in vivo (1, 5), but they are in vivo substrates for N-3 methylation (15). Escherichia coli K12 strains (designated mec+) also specify a DNA-cytosine methylase; and it was observed that after growth in mec+ cells, phage X is only weakly restricted by strains harboring the N-3 factor. In contrast, when X is grown in strains which lack cytosine methylase activity (designated mec- [17, 22]),
it is subject to strong restriction by N-3-containing cells (17). Thus, it appeared that the K12 cytosine-methylase partially protects phage A DNA against N-3 restriction. A previous report from this laboratory demonstrated that in vivo, the N-3 modification enzyme methylates approximately twice as many cytosine residues on A or fd DNA as does the host K12 mec+ cytosine methylase (17). These observations led to the question of whether the K12 and N-3 methylases methylate some common sequence(s) on A or fd phage DNAs. To explore these possibilities and to obtain more direct data on the nature of the methylation sites, we have analyzed the distribution of 5-methylcytosine (MeC) in pyrimidine tracts derived from isotopically labeled viral DNAs propagated in various host cells; furthermore, we have analyzed the extents of in vitro methylation of various A DNAs. The results presented here demonstrate a striking similarity in the K12 and N-3specified methylation patterns. We propose that the two enzymes methylate the same sites in vitro, and that the differences observed for the in vivo methylation levels may be due to differences in the respective rates of methylation.
129
130
MATERIALS AND METHODS
Phage and bacterial strains. Phage X c1857 indgenerously provided by B. Dottin; phage fd was from D. Marvin. E. coli 1100 sup+ end I- thi- mK+ was obtained from H. Revel and made lysogenic for A c1857 and/or recipient of an F+ factor. From this strain the mec- mutant lacking DNA-cytosine methylase was isolated (17). E. coli B is strain B834 met- gal- rB-mB- (28). In vivo methylation of DNA in phages fd and A. Media and conditions of labelling of the methylated bases in the presence of L-[methyl-3H]methionine, phage purification and isolation of DNA were essentially as described earlier (14, 15). In vitro methylation of calf thymus DNA. Calf thymus DNA was methylated in vitro with a crude extract of E. coli B (N-3) cells by using S-adenosyl-L[methyl-3H]methionine as the methyl donor. The reaction mixture and procedure for isolation of the labeled DNA were described earlier (14). Depurination of DNA. DNA was degraded by diphenylamine in acid solution to yield free purines, inorganic phosphate, and pyrimidine oligonucleotides of the general formula Pynpn2 l (Py, pyrimidine deoxynucleoside; and p, phosphate [9]). Calf thymus DNA (75 to 100 mg; Worthington) and 20,000 to 300,000 counts/min of [3H-methyl]-labeled DNA were dissolved in 25 ml of water. 90.5% Formic acid (72 ml; Baker) containing 2 g of (Eastman) diphenylamine was added, and the mixture was heated at 32 C for 18 h in the dark. Diphenylamine and formic acid were removed by the method of Spencer et al. (26). Separation of pyrimidine oligonucleotides according to chain length. The solution (approximately 80 ml) of depurinated DNA was made 7 M in urea (Mallinckrodt) and applied at room temperature to a column (30 by 1 cm) of diethylaminoethyl (DEAE)-cellulose (Schleicher & Schuell) equilibrated in 7 M urea-0.1 M sodium acetate, pH 5.5. The column was washed extensively until free purines, inorganic phosphate, and monopyrimidine diphosphates were eluted. Pyrimidine oligonucleotides of similar chain length (isostichs) were separated according to size by elution with a linear gradient (800 ml total) of 0 to 0.25 M or 0.3 M NaCl in 7 M urea0.1 M sodium acetate, pH 5.5 (19). Fractions of 2.6 ml were collected and their values for absorbance at 260 nm and radioactivity were determined. When isostich no. 8 had been eluted, the column was then washed with 1 M NaCl in 7 M urea-0.1 M sodium acetate buffer, pH 5.5, to remove larger isostichs. Most (90 to 100%) of the input radioactivity was recovered. NaCl concentrations (see Fig. 1 and 3) represent calculated values based on the number of fractions collected, volume per fraction, and total volume per gradient. Fractionation of isostichs according to base composition. The pooled peak fractions corresponding to each isostich were diluted threefold with water and applied to a column (25 by 0.5 cm) of DEAE cellulose in 7 M urea-0.1 M sodium acetate, pH 5.5. The column was washed with about 30 ml of 7 M urea-0.1 M formic acid (pH 3.5) and then eluted was
J. BACTERIOL.
MAY AND HATTMAN
with a 200- or 300-ml linear gradient of NaCl in 7 M urea-0.1 M formic acid, pH 3.5. The NaCl gradient concentration used depended on the chain length of the particular isostich. The order of elution of compositional isomers varies with thymine content; e.g., for the isostich of chain length equal to 3, the order of elution is C3, C2T, CT2, and T3 (24; C, cytosine, T, thymine). The NaCl concentrations (see Fig. 2 and 4) were calculated in a fashion similar to that described above. Desalting of column fractions. Peak fractions containing the various compositional isomers were pooled, diluted fivefold with water and applied to columns (0.8 by 3 cm) of DEAE cellulose (carbonate form) packed under pressure. Each column was washed with 25 ml of 0.005 M (NH4)2C03 to remove urea and salts. Pyrimidine tracts were eluted with 10 ml of 1 M (NH4)2CO3. The (NH4)2CO3 was removed by repeatedly adding water and evaporating samples to dryness in vacuo over P205. Preparation of cell-free extracts. E. coli F+ 1100 strains mec+ and mec- (N-3) were grown overnight in broth; the plasmid-containing cells were grown in the presence of either tetracycline (20 Ag/ml) or streptomycin (40 ,ug/ml). The cells were diluted 50-fold into 500 ml of fresh, drug-free medium, incubated with aeration at 37 C for 3.5 h (ca. 4 x 103 cells/ml), and harvested by centrifugation. The cells were suspended in 5 ml of 0.01 M postassium phosphate buffer, pH 7.2, 0.005 M MgCl2, 0.01 M 2-mercaptoethanol, and 0.01 M disodium ethylenediaminetetraacetic acid (EDTA) and then disrupted by sonication. After clarification by centrifugation for 30 min at 20,000 x g, the extract was incubated for 1 h at 37 C in the presence of 20 yg of pancreatic DNase per ml. The extract was then centrifuged for 1 h at 170,000 x g in an IEC-A-321 rotor. The supernatant fluid was collected and dialyzed against 0.01 M potassium phosphate buffer, pH 7.2, 5% glycerol (wt/vol), 0.001 M 2-mercaptoethanol, 0.025 M NaCl, and 0.01 M disodium EDTA. The dialyzed cell-free extract was stored at 5 C. Methylation of bacteriophage A DNA in vitro. Phage A DNA was obtained from purified phage after propagation in E. coli B rB-mB- mec- or E. coli K12 mec+ (P1); the former DNA lacks MeC. The in vitro methylation reaction mixtures (1 ml) contained the following: 25 to 70 Mg of A DNA, 200 to 400 Mg of extract protein, 80 gmol of Tris(hydroxymethyl)aminomethane-hydrochloride, pH 7.3, 8 umol of disodium EDTA, pH 7.0, 10 umol of 2-mercaptoethanol, 3.4 nmol of (3.6
x
105 counts/min) of S-
adenosy-L-[methyl- 4C]methionine. At appropriate intervals, samples were put into cold 5% trichloroacetic acid, and the acid-insoluble fraction was collected on to GF/A glass-fiber filter disks. After 2.5 to 3 h of incubation, when the methylation reaction was complete, the remainder of the incubation mixture was deproteinized by phenol-extraction (with 40 Mg of carrier calf thymus DNA), and nucleic acids were precipitated with ethanol at -20 C overnight. After centrifugation at 15,000 x g for 30 min, the resulting pellets were suspended in 1 N NaOH and incubated 2 h at 37 C. The solutions were neutralized and then made 5% with perchloric acid. The precipitates were
VOL. 122, 1975
DNA-CYTOSINE METHYLASES
subjected to alternate cycles of centrifugation and washing in 5% perchloric acid and 95% ethanol. The DNA pellet was dried and dissolved in 0.1 ml 70% perchloric acid and hydrolyzed for 1 h in a boiling water bath. Chromatographic analysis of the radioactively labeled methylated bases was as described previously (14). It should be noted that control assays
lacking added DNA were run in parallel; the amount of label retained on these filter disks was less than 10% that observed in the reaction mixtures containing DNA after the methylation reaction ceased due to saturation.
RESULTS
Analysis of MeC-containing pyrimidine X DNA. DNA was isolated from phage X after propagation in the presence of L-[methyl-3H]methionine in E. coli B mece (N-3) and in E. coli K12 mec+ host cells. After depurination, the resulting oligopyrimidine tracts were separated according to chain length (see Materials and Methods). The results of a typical chromatographic profile are shown in Fig. 1 for X DNA obtained from phage grown in E. coli B mec- (N-3) cells. The purines do not bind to DEAE-cellulose in the presence of 7 M urea and pass through the column (19, 23). The radioactive label observed in these fractions is tracts from
131
presumably the N6-methyladenine present in A DNA (Fig. 1). After continued washing with the buffer used to equilibrate the column, a second peak of ultraviolet absorbing material is eluted which contains no 3H-label; chromatographic analysis of a perchloric acid hydrolysate showed that this peak contained only cytosine and thymine. Other workers have shown that the first pyrimidine isostich is eluted by the column buffer (19). The salt gradient was applied after the first pyrimidine isostich peak had been eluted. It is clear (Fig. 1) that the majority of [3HIMeC occurs in isostich 3 (namely, Py3p4). The skewing of 3H-label versus the absorbance profile is often seen; it is presumably due to partial separation of the cytosine-rich compositional isomers (23). The distribution of [3H]MeC in pyrimidine tracts from A-K mec+ DNA was virtually identical to that shown for the tracts from A-B mec- (N-3) DNA. The labeled isostich peaks were then rechromatographed at pH 3.5 to afford resolution into compositional isomers of identical cytosine and thymine content (see Materials and Methods). Profiles of rechromatographed isostichs 2 through 5 from A*B mec- (N-3) DNA are depicted in Fig. 2. It is evident that MeC is not
600-
>8
100-
500-
IM-NoCI 400l:
75>8~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
50Pu
V. 25-
r t .L.~~~~~~~~~~~~ \6
100-
6
,..~~~~
0
7
&mm
o I.I
-
0-
-T W--
50
)F0
150
200
r-
1. I--
A"& AM .,. r=
250
FRACTION NUMBER
FIG. 1. Chromatography of pyrimidine tracts from depurinated 3H-X B mec- (N-3) DNA and carrier calf thymus DNA. Depurinated 3H-labeled A B mec- (N-3) DNA (- 20,000 counts/min) and depurinated carrier calf thymus DNA (100 mg) were co-chromatographed on a column (30 by 1 cm) of DEAE cellulose in 7 M ureaand 0.1 M sodium acetate, pH 5.5. The column was washed extensively with 7 M urea-0.1 M sodium acetate (pH 5.5) and then eluted with a linear gradient of 0 to 0.3 M NaCI in 7 M urea-0.1 M sodium acetate (pH 5.5) until pyrimidine tract no. 8 was eluted. The column was then washed with 1 M NaCI in 7 M urea-0.1 M sodium acetate (pH 5.5) to elute large fragments. Fraction numbers I to 110 were 5.2 ml each; the gradient fractions were 2.6 ml each. The continuous line represents the absorbance at 260 nm; the dotted line represents the calculated NaCI concentration. 0, 3H-label counts per minute per 0.2 ml.
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132
02
NK
-0.1
0
0
80
20
40
FRACTION NUMBER FIG. 2. Rechromatography of 3H-labeled isostichs at pH 3.5. 3H-containing isostichs derived from A B mec(N-3) DNA (Fig. 1) were rechromatographed on columns (25 by 0.5 cm) of DEAE cellulose in 7 M urea-0.1 M formic acid, pH 3.5. Elution was carried out by a linear gradient from 0 to 0.075, 0.10, 0.15, or 0,20 M NaCl (in 7 M urea-0.1 M formic acid, pH 3.5) for isostichs 2, 3, 4,
or
5, respectively. No additional 3H-label or
ultraviolet-absorbing material was eluted with 1 M NaCI (in 7 M urea-0.1 M formic acid). The graphs plotted as in Fig. 1. Fractions of 2.6 ml were collected.
found in all the cytosine-containing isomers of a given isostich; e.g., in isostich 3, there is relatively little MeC in the C3 and CT2 isomers, whereas almost all the MeC is in the C2T isomer (Fig. 2b). Several of the isomers were checked for base composition by perchloric acid hydrolysis, paper chromatography, and spectrophotometric analysis; the results were consistent with the designated compositions (data not shown). Table 1 contains a summary of the data obtained for both A-B mec- (N-3) and A-K mec+ DNA. Listed in the table are the percentages of total radioactive MeC label observed in each pyrimidine isostich, as well as among the compositional isomers of isostichs 2 through 5. The distribution of MeC produced by host- and plasmid-specific methylation of A DNA is strikingly similar. For example, in each case the major MeC-containing pyrimidine isostich must have been derived from a site on A DNA of the sequence ... Pu(pCpCpTp)Pu...; the parenthesis indicates that the pyrimidine sequence is
are
not yet known. For both methylases, no methylation occurs at a cytosine residue located between two purines, since no MeC is observed in
isostich 1. Analysis of MeC-containing pyrimidine tracts from single-stranded fd DNA. The single-stranded DNA obtained from mature fd phage grown in K12 mec+ cells was observed to contain on the average 1.5 MeC per singlestranded DNA molecule (15, 17). When fd is grown in mec+ (N-3) or in mec- (N-3) cells, three MeC residues per fd DNA molecule are observed. Since there are relatively few MeC residues per DNA molecule, it was of special interest to determine in which pyrimidine tract(s) they occur and to compare host- versus plasmid-specific methylation. When labeled fd DNA was analyzed by the method used for X DNA in the previous section, it was found that the MeC distribution was much simpler. All of the 3H-MeC label was observed in isostich 3 (Fig 3); furthermore, almost all of this label is found in the C2T
133
DNA-CYTOSINE METHYLASES
VOL. 122, 1975
TBmLE 1. Distribution of [3HJ5-MeC in pyrimidine tracts isolated from in vivo labeled A * B mec- (N-3) DNA and X-K-12 mec+ DNA Total ['HJMeCa (%) for phage: A K mec+ A* imec- (N-3)
Pyrimidine isostich no.
1
0
0
2
6.6
5.0
Compositional isomer
C2
CT 65.9
3
13.2
4
64.8
16.2
7.0
A K mec+
6.2 0.4
4.9 0.1
CS
3.0
CT2
62.4 0.5
7.4 57.4 0
C4 CST
0 3.4 9.8
1.1 5.4 9.7
C3T2 C2T3
0.1 1.1 5.8
0.1 0.9 4.5
C4T
5.5
A*B mec- (N-3)
C2T
C2T2 5
Total [3H]MeCa
6
1.6
1.8
NA
NA
NA
7
5.6
4.5
NA
NA
NA
>7
8
20
400
15-
(N~~~~~~~~~~~~~~~~
OFRACTION NUMER
FIG. 3. Chromatography of pyrimidine tracts from depurinated 3H-fd-mec- (N-3) DNA. Depurinated, 3H-labeled fd mec- (N-3) DNA (- 30,000 counts/min) and depurinated carrier calf thymus DNA (75 mg) were co-chromatographed on DEAE celMlose in 7 M urea-0.1 M sodium acetate, pH 5.5. The column was washed extensively with 7 M urea-0.1 M sodium acetate (pH 5.5) and then eluted with a linear gradient of 0 to 0.25 M NaCI in 7 M urea-0.1 M sodium acetate (pH 5.5) until pyrimidine tract no. 8 was eluted. The column was then washed with 1 M NaCI in 7 M urea-0.1 M acetate (pH 5.5). Fraction numbers 1 to 100 were 5.2 ml each; the gradient fractions were 2.6 ml each. The graphs are plotted as in Fig. 1. -
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plasmid methylase. For this purpose, calf thymus DNA was methylated with a crude extract derived from B mec (N-3) cells in the presence of S-adenosyl-L- [methyl-3H ]-methionine, and the DNA was subjected to depurination and pyrimidine tract analysis (see Materials and Methods). The distribution of MeC among the various pyrimidine isostichs and their compositional isomers is summarized in Table 3. The MeC distribution in calf thymus DNA pyrimidine tracts was similar to that for X DNA (see Table 1), although some differences in the relative labeling of' isostichs 2 and 3 were observed. Methylation of X DNA in vitro: saturation levels of methylated bases. The data presented above reveal a striking similarity in the distribution of MeC in various pyrimidine tracts. These results were unexpected, especially in view of the twofold increase in MeC -1000 content for X or fd DNA propagated in mecF.CZ (N-3) versus mec+ hosts. If the additional MeC lzz.l produced by N-3 were in a new sequence(s), we c might have anticipated a change in the MeC 4I-i :50 distribution among pyrimidine tracts. However, -500 if on the other hand, the MeC levels that are produced on X and f'd DNAs reflect a difference in the in vivo rate of methylation or availability ";:k of' sites prior to phage DNA maturation, then we should expect that A DNA would be methylated in vitro to the same extent by the K12 mec+ and N-3 methylases. This possibility was tested by 100 20 80 0 40 60 using cell-free extracts derived from K12 mec+ FRACTION NUMBER FIG. 4. Rechromatography of 3H-labeled isostich and K12 mec- (N-3) cultures (see Materials and no. 3 from fd.mec- (N-3) DNA. The 3H-containing Methods). The results summarized in Table 4 demonisostich (Fig. 3) was rechromatographed on a column (25 by 0.5 cm) of DEAE cellulose in 7 M urea-0.1 M strate that the mec+ extract is capable of formic acid (pH 3.5), as in Fig. 2. Fractions were 2.6 methylating various A DNAs in vitro to an ml each. extent similar to that observed with the mec-
isomer (Fig 4). This result is the same for fd-mec+, fd-mec+ (N-3), and fd-mec- (N-3) phage DNA (Table 2). We should point out that C2T is also the major MeC-containing isomer in A DNA. Analysis of MeC-containing pyrimidine tracts from calf thymus DNA methylated in vitro by a crude extract of E. coli B mec(N-3) cells. Calf thymus DNA has a base composition (44% GC) significantly different than A DNA (50% GC); in addition, due to the large amount of thymus DNA per unit genome, it contains a greater possible variety of nucleotide sequences than A DNA. Thus, we were interested to see how these factors affect the distribution of MeC residues produced by the
TABLE 2. Distribution of [3H]5-MeC in pyrimidine tracts isolated from in viuo labeled fd mec- (N-3), fd. mec+ and fd mec+ (N-3) DNA
Pyrimidine fd DNA
isostich no.
fd
mec-
(N-3)
3
3Total THoMeCa ~~(%)
100
.Total
Copstoa
Compositional ismr C3 C 2T
CT2 fd mec+
fd.mec- (N-3)
3
3
100
100
[3H]MeC % 8
92 0
C3
1
C2T CT2
99 0
C33
15 85
C2T
a The data for fd mec- (N-3) DNA are taken from Figs. 3 and 4. The data for fd mec+ and fd mec+ (N-3) were obtained from separate experiments.
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DNA-CYTOSINE METHYLASES
(N-3) extract. It is interesting to note that A * K mec+ DNA, which already contained approximately 65 MeC per DNA, is methylated to similar extents by both mec+ and mec- (N-3) extracts. Thus, in vitro the mec+ and N-3 methylases appear to recognize the same sites on X DNA. One unexplained discrepancy in the data must be mentioned; namely, the calculated number of MeC per X DNA is about one-half that observed for in vivo labeled XA mec- (N-3) DNA (extracted from mature virus particles). We know that the data summarized in Table 4 were obtained from DNA methylated to saturation. This was verified independently in the following manner. When comparable assay mixtures were prepared and "4C-methyl label incorporation attained its plateau value, addition of either mec+ or mec(N-3) extract had no effect on this value, but addition of more DNA led to resumption of incorporation up to a level directly proportional to the amount of added DNA. Therefore, the observed extents of methylation are not artifacts of enzyme inhibition (inactivation) during the in vitro incubation. DISCUSSION In this communication we have analyzed the distribution of [3H]5-methylcytosine among pyrimidine tracts for phage propagated on various host cells. The distribution of MeC among pyrimidine tracts was found to be the same for phage DNA labeled in E. coli K12 mec+ versus E. coli K12 mec- (N-3) bacteria. These results are surprising in view of the twofold higher MeC content for phage grown in N-3 containing cells (17). However, two alternative models can account for the results. In the first model, the N-3 enzyme recognizes the K12 mec+ sequence(s) and a new related sequence(s) differing from the former either in the nature of the purine residues outside the pyrimidine runs, or in the pyrimidine sequence within the runs. This notion can be illustrated for the sole MeC-containing tract in fd DNA, namely, C2T. This tract must originate from a site on the DNA containing the sequence The difference NpPu (pCpCpTp)PupN between the K12 mec+ and the N-3 methylation recognition could reside at the proximal purine (Pu) residues (or distal bases N etc.) or the difference might lie within the tripyrimidine ...
...
135
TABLE 3. Distribution of [3H]5-MeC in pyrimidine tracts isolated from in vitro labeled calf thymus DNA Pyrimidine isostich no.
Total 3H-MeCa (%)
Compositional isomer
Total sH-MeCa (%)
1
7
3.1
NA
NA
The data were obtained from experiments analogous to those for A and fd DNA. NA, not analyzed. a
methylation in mec+ versus mec- (N-3) host cells. The lower phage MeC content after growth in mec+ cells may be due to differences in the mec+ versus (N-3) enzymes, or it could reflect a lower methylase concentration in mec+ cells. Several independent observations support the second model. Firstly, nothwithstanding a twofold difference in MeC content, we observed identical distributions of [3H]MeC among pyrimidine isostichs for phage grown in mec(N-3) and mec+ cells (Tables 1 and 2). Recent experiments also show that the methylated tripyrimidine sequence is C-MeC-T for both fd-mec+ and fd-mec- (N-3) DNAs (May and Hattman, manuscript in preparation). Secondly, the in vitro methylation experiments reveal that under saturation conditions the mec- (N-3) and mec+ extracts methylate cyto-
sine residues on A DNA to the same extent (Table 4). Although the MeC level produced in vitro is somewhat lower than expected, it is clear that the two methylases modify the same sequence. The second model proposes that the N-3 and number of sites. This situation is consistent K12 mec+ enzymes recognize identical se- with the second model only. Finally, only a small difference in MeC content (< 20%) was quences but that the difference in MeC levels on phage DNAs is due to a lower in vivo rate of observed for host E. coli DNA isolated from
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TABLE 4. In vitro methylation of phage X DNA by crude extracts of mec+ and mec- (N-3) cellsa Calculatedd Expt. Extrt Observedc Acceptorb Calculatedd xtrac no. DNA MeC/MeAde ['4CJMec/DNA [4C]MeAde/DNA 1
XABmecXAKmec+
2
XABmecX B mec- (N-3)
4.6
82
18
81 50
mec- (N-3)
4.3 3.3 3.8
51
19 15 14
mec+ mec- (N-3) mec+ mec- (N-3)
3.5 3.9 0.06 0.03
94 99
Vol. 122, No. 1 Printed in U.S.A.
Deoxyribonucleic Acid-Cytosine Methylation by Host- and Plasmid-Controlled Enzymes MAUREEN S. MAY AND STANLEY HAFTMAN*
University of Rochester, Department of Biology, Rochester, New York
14627
Received for publication 17 January 1975
Deoxyribonucleic acid (DNA)-cytosine methylation specified by the wild-type Escherichia coli K12 mec+ gene and by the N-3 drug resistance (R) factor was studied in vivo and in vitro. Phage A and fd were propagated in the presence of L- [methyl-'H]methionine in various host bacteria. The in vivo labeled DNA was isolated from purified phage and depurinated by formic acid-diphenylamine treatment. The resulting pyrimidine oligonucleotide tracts were separated according to size and base composition by chromatography on diethylaminoethyl-cellulose in 7 M urea at pH 5.5 and 3.5, respectively. The distribution of labeled 5-methylcytosine in DNA pyrimidine tracts was identical for phage grown in mec+ and mec- (N-3) cells. For phage A the major 5-methylcytosinecontaining tract was the tripyrimidine, C2T; for both fd- mec- (N-3) DNA and fd mec+ DNA, C2T was the sole 5-methylcytosine-containing tract. When various A DNAs were methylated to saturation in vitro by crude extracts from mec+ and mec- (N-3) cells, the extent of cytosine methylation was the same. This is in contrast to in vivo methylation where A mec (N-3) DNA contains twice as many 5-methylcytosines per genome as A mec+ DNA. Therefore, we suggest that the K12 met+ cytosine methylase and the N-3 plasmid modification methylase are capable of recognizing the same nucleotide sequences, but that the in vivo methylation rate is lower in mec+ cells.
Many bacterial strains and some plasmids control deoxyribonucleic acid (DNA) restriction modification systems (2, 7). These systems enable a strain to protect its own DNA while foreign DNA is degraded. Modification is brought about by an adenine- or cytosinemethylase which methylates specific sequences on double-stranded DNA. Restriction occurs via an endonuclease which cleaves unmodified double-stranded DNA. The N-3 plasmid, a fi- drug resistance factor related to the R-15 plasmid, controls hspH host specificity (3, 4, 27, 29). This host specificity system is based on DNA-cytosine methylation (16, 25). The N-3 plasmid can restrict and/or modify double-stranded DNA phages, including coliphage X. The singlestranded DNA phages, fd and M13, are not subject to N-3 restriction in vivo (1, 5), but they are in vivo substrates for N-3 methylation (15). Escherichia coli K12 strains (designated mec+) also specify a DNA-cytosine methylase; and it was observed that after growth in mec+ cells, phage X is only weakly restricted by strains harboring the N-3 factor. In contrast, when X is grown in strains which lack cytosine methylase activity (designated mec- [17, 22]),
it is subject to strong restriction by N-3-containing cells (17). Thus, it appeared that the K12 cytosine-methylase partially protects phage A DNA against N-3 restriction. A previous report from this laboratory demonstrated that in vivo, the N-3 modification enzyme methylates approximately twice as many cytosine residues on A or fd DNA as does the host K12 mec+ cytosine methylase (17). These observations led to the question of whether the K12 and N-3 methylases methylate some common sequence(s) on A or fd phage DNAs. To explore these possibilities and to obtain more direct data on the nature of the methylation sites, we have analyzed the distribution of 5-methylcytosine (MeC) in pyrimidine tracts derived from isotopically labeled viral DNAs propagated in various host cells; furthermore, we have analyzed the extents of in vitro methylation of various A DNAs. The results presented here demonstrate a striking similarity in the K12 and N-3specified methylation patterns. We propose that the two enzymes methylate the same sites in vitro, and that the differences observed for the in vivo methylation levels may be due to differences in the respective rates of methylation.
129
130
MATERIALS AND METHODS
Phage and bacterial strains. Phage X c1857 indgenerously provided by B. Dottin; phage fd was from D. Marvin. E. coli 1100 sup+ end I- thi- mK+ was obtained from H. Revel and made lysogenic for A c1857 and/or recipient of an F+ factor. From this strain the mec- mutant lacking DNA-cytosine methylase was isolated (17). E. coli B is strain B834 met- gal- rB-mB- (28). In vivo methylation of DNA in phages fd and A. Media and conditions of labelling of the methylated bases in the presence of L-[methyl-3H]methionine, phage purification and isolation of DNA were essentially as described earlier (14, 15). In vitro methylation of calf thymus DNA. Calf thymus DNA was methylated in vitro with a crude extract of E. coli B (N-3) cells by using S-adenosyl-L[methyl-3H]methionine as the methyl donor. The reaction mixture and procedure for isolation of the labeled DNA were described earlier (14). Depurination of DNA. DNA was degraded by diphenylamine in acid solution to yield free purines, inorganic phosphate, and pyrimidine oligonucleotides of the general formula Pynpn2 l (Py, pyrimidine deoxynucleoside; and p, phosphate [9]). Calf thymus DNA (75 to 100 mg; Worthington) and 20,000 to 300,000 counts/min of [3H-methyl]-labeled DNA were dissolved in 25 ml of water. 90.5% Formic acid (72 ml; Baker) containing 2 g of (Eastman) diphenylamine was added, and the mixture was heated at 32 C for 18 h in the dark. Diphenylamine and formic acid were removed by the method of Spencer et al. (26). Separation of pyrimidine oligonucleotides according to chain length. The solution (approximately 80 ml) of depurinated DNA was made 7 M in urea (Mallinckrodt) and applied at room temperature to a column (30 by 1 cm) of diethylaminoethyl (DEAE)-cellulose (Schleicher & Schuell) equilibrated in 7 M urea-0.1 M sodium acetate, pH 5.5. The column was washed extensively until free purines, inorganic phosphate, and monopyrimidine diphosphates were eluted. Pyrimidine oligonucleotides of similar chain length (isostichs) were separated according to size by elution with a linear gradient (800 ml total) of 0 to 0.25 M or 0.3 M NaCl in 7 M urea0.1 M sodium acetate, pH 5.5 (19). Fractions of 2.6 ml were collected and their values for absorbance at 260 nm and radioactivity were determined. When isostich no. 8 had been eluted, the column was then washed with 1 M NaCl in 7 M urea-0.1 M sodium acetate buffer, pH 5.5, to remove larger isostichs. Most (90 to 100%) of the input radioactivity was recovered. NaCl concentrations (see Fig. 1 and 3) represent calculated values based on the number of fractions collected, volume per fraction, and total volume per gradient. Fractionation of isostichs according to base composition. The pooled peak fractions corresponding to each isostich were diluted threefold with water and applied to a column (25 by 0.5 cm) of DEAE cellulose in 7 M urea-0.1 M sodium acetate, pH 5.5. The column was washed with about 30 ml of 7 M urea-0.1 M formic acid (pH 3.5) and then eluted was
J. BACTERIOL.
MAY AND HATTMAN
with a 200- or 300-ml linear gradient of NaCl in 7 M urea-0.1 M formic acid, pH 3.5. The NaCl gradient concentration used depended on the chain length of the particular isostich. The order of elution of compositional isomers varies with thymine content; e.g., for the isostich of chain length equal to 3, the order of elution is C3, C2T, CT2, and T3 (24; C, cytosine, T, thymine). The NaCl concentrations (see Fig. 2 and 4) were calculated in a fashion similar to that described above. Desalting of column fractions. Peak fractions containing the various compositional isomers were pooled, diluted fivefold with water and applied to columns (0.8 by 3 cm) of DEAE cellulose (carbonate form) packed under pressure. Each column was washed with 25 ml of 0.005 M (NH4)2C03 to remove urea and salts. Pyrimidine tracts were eluted with 10 ml of 1 M (NH4)2CO3. The (NH4)2CO3 was removed by repeatedly adding water and evaporating samples to dryness in vacuo over P205. Preparation of cell-free extracts. E. coli F+ 1100 strains mec+ and mec- (N-3) were grown overnight in broth; the plasmid-containing cells were grown in the presence of either tetracycline (20 Ag/ml) or streptomycin (40 ,ug/ml). The cells were diluted 50-fold into 500 ml of fresh, drug-free medium, incubated with aeration at 37 C for 3.5 h (ca. 4 x 103 cells/ml), and harvested by centrifugation. The cells were suspended in 5 ml of 0.01 M postassium phosphate buffer, pH 7.2, 0.005 M MgCl2, 0.01 M 2-mercaptoethanol, and 0.01 M disodium ethylenediaminetetraacetic acid (EDTA) and then disrupted by sonication. After clarification by centrifugation for 30 min at 20,000 x g, the extract was incubated for 1 h at 37 C in the presence of 20 yg of pancreatic DNase per ml. The extract was then centrifuged for 1 h at 170,000 x g in an IEC-A-321 rotor. The supernatant fluid was collected and dialyzed against 0.01 M potassium phosphate buffer, pH 7.2, 5% glycerol (wt/vol), 0.001 M 2-mercaptoethanol, 0.025 M NaCl, and 0.01 M disodium EDTA. The dialyzed cell-free extract was stored at 5 C. Methylation of bacteriophage A DNA in vitro. Phage A DNA was obtained from purified phage after propagation in E. coli B rB-mB- mec- or E. coli K12 mec+ (P1); the former DNA lacks MeC. The in vitro methylation reaction mixtures (1 ml) contained the following: 25 to 70 Mg of A DNA, 200 to 400 Mg of extract protein, 80 gmol of Tris(hydroxymethyl)aminomethane-hydrochloride, pH 7.3, 8 umol of disodium EDTA, pH 7.0, 10 umol of 2-mercaptoethanol, 3.4 nmol of (3.6
x
105 counts/min) of S-
adenosy-L-[methyl- 4C]methionine. At appropriate intervals, samples were put into cold 5% trichloroacetic acid, and the acid-insoluble fraction was collected on to GF/A glass-fiber filter disks. After 2.5 to 3 h of incubation, when the methylation reaction was complete, the remainder of the incubation mixture was deproteinized by phenol-extraction (with 40 Mg of carrier calf thymus DNA), and nucleic acids were precipitated with ethanol at -20 C overnight. After centrifugation at 15,000 x g for 30 min, the resulting pellets were suspended in 1 N NaOH and incubated 2 h at 37 C. The solutions were neutralized and then made 5% with perchloric acid. The precipitates were
VOL. 122, 1975
DNA-CYTOSINE METHYLASES
subjected to alternate cycles of centrifugation and washing in 5% perchloric acid and 95% ethanol. The DNA pellet was dried and dissolved in 0.1 ml 70% perchloric acid and hydrolyzed for 1 h in a boiling water bath. Chromatographic analysis of the radioactively labeled methylated bases was as described previously (14). It should be noted that control assays
lacking added DNA were run in parallel; the amount of label retained on these filter disks was less than 10% that observed in the reaction mixtures containing DNA after the methylation reaction ceased due to saturation.
RESULTS
Analysis of MeC-containing pyrimidine X DNA. DNA was isolated from phage X after propagation in the presence of L-[methyl-3H]methionine in E. coli B mece (N-3) and in E. coli K12 mec+ host cells. After depurination, the resulting oligopyrimidine tracts were separated according to chain length (see Materials and Methods). The results of a typical chromatographic profile are shown in Fig. 1 for X DNA obtained from phage grown in E. coli B mec- (N-3) cells. The purines do not bind to DEAE-cellulose in the presence of 7 M urea and pass through the column (19, 23). The radioactive label observed in these fractions is tracts from
131
presumably the N6-methyladenine present in A DNA (Fig. 1). After continued washing with the buffer used to equilibrate the column, a second peak of ultraviolet absorbing material is eluted which contains no 3H-label; chromatographic analysis of a perchloric acid hydrolysate showed that this peak contained only cytosine and thymine. Other workers have shown that the first pyrimidine isostich is eluted by the column buffer (19). The salt gradient was applied after the first pyrimidine isostich peak had been eluted. It is clear (Fig. 1) that the majority of [3HIMeC occurs in isostich 3 (namely, Py3p4). The skewing of 3H-label versus the absorbance profile is often seen; it is presumably due to partial separation of the cytosine-rich compositional isomers (23). The distribution of [3H]MeC in pyrimidine tracts from A-K mec+ DNA was virtually identical to that shown for the tracts from A-B mec- (N-3) DNA. The labeled isostich peaks were then rechromatographed at pH 3.5 to afford resolution into compositional isomers of identical cytosine and thymine content (see Materials and Methods). Profiles of rechromatographed isostichs 2 through 5 from A*B mec- (N-3) DNA are depicted in Fig. 2. It is evident that MeC is not
600-
>8
100-
500-
IM-NoCI 400l:
75>8~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
50Pu
V. 25-
r t .L.~~~~~~~~~~~~ \6
100-
6
,..~~~~
0
7
&mm
o I.I
-
0-
-T W--
50
)F0
150
200
r-
1. I--
A"& AM .,. r=
250
FRACTION NUMBER
FIG. 1. Chromatography of pyrimidine tracts from depurinated 3H-X B mec- (N-3) DNA and carrier calf thymus DNA. Depurinated 3H-labeled A B mec- (N-3) DNA (- 20,000 counts/min) and depurinated carrier calf thymus DNA (100 mg) were co-chromatographed on a column (30 by 1 cm) of DEAE cellulose in 7 M ureaand 0.1 M sodium acetate, pH 5.5. The column was washed extensively with 7 M urea-0.1 M sodium acetate (pH 5.5) and then eluted with a linear gradient of 0 to 0.3 M NaCI in 7 M urea-0.1 M sodium acetate (pH 5.5) until pyrimidine tract no. 8 was eluted. The column was then washed with 1 M NaCI in 7 M urea-0.1 M sodium acetate (pH 5.5) to elute large fragments. Fraction numbers I to 110 were 5.2 ml each; the gradient fractions were 2.6 ml each. The continuous line represents the absorbance at 260 nm; the dotted line represents the calculated NaCI concentration. 0, 3H-label counts per minute per 0.2 ml.
J. BACTERIOL.
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132
02
NK
-0.1
0
0
80
20
40
FRACTION NUMBER FIG. 2. Rechromatography of 3H-labeled isostichs at pH 3.5. 3H-containing isostichs derived from A B mec(N-3) DNA (Fig. 1) were rechromatographed on columns (25 by 0.5 cm) of DEAE cellulose in 7 M urea-0.1 M formic acid, pH 3.5. Elution was carried out by a linear gradient from 0 to 0.075, 0.10, 0.15, or 0,20 M NaCl (in 7 M urea-0.1 M formic acid, pH 3.5) for isostichs 2, 3, 4,
or
5, respectively. No additional 3H-label or
ultraviolet-absorbing material was eluted with 1 M NaCI (in 7 M urea-0.1 M formic acid). The graphs plotted as in Fig. 1. Fractions of 2.6 ml were collected.
found in all the cytosine-containing isomers of a given isostich; e.g., in isostich 3, there is relatively little MeC in the C3 and CT2 isomers, whereas almost all the MeC is in the C2T isomer (Fig. 2b). Several of the isomers were checked for base composition by perchloric acid hydrolysis, paper chromatography, and spectrophotometric analysis; the results were consistent with the designated compositions (data not shown). Table 1 contains a summary of the data obtained for both A-B mec- (N-3) and A-K mec+ DNA. Listed in the table are the percentages of total radioactive MeC label observed in each pyrimidine isostich, as well as among the compositional isomers of isostichs 2 through 5. The distribution of MeC produced by host- and plasmid-specific methylation of A DNA is strikingly similar. For example, in each case the major MeC-containing pyrimidine isostich must have been derived from a site on A DNA of the sequence ... Pu(pCpCpTp)Pu...; the parenthesis indicates that the pyrimidine sequence is
are
not yet known. For both methylases, no methylation occurs at a cytosine residue located between two purines, since no MeC is observed in
isostich 1. Analysis of MeC-containing pyrimidine tracts from single-stranded fd DNA. The single-stranded DNA obtained from mature fd phage grown in K12 mec+ cells was observed to contain on the average 1.5 MeC per singlestranded DNA molecule (15, 17). When fd is grown in mec+ (N-3) or in mec- (N-3) cells, three MeC residues per fd DNA molecule are observed. Since there are relatively few MeC residues per DNA molecule, it was of special interest to determine in which pyrimidine tract(s) they occur and to compare host- versus plasmid-specific methylation. When labeled fd DNA was analyzed by the method used for X DNA in the previous section, it was found that the MeC distribution was much simpler. All of the 3H-MeC label was observed in isostich 3 (Fig 3); furthermore, almost all of this label is found in the C2T
133
DNA-CYTOSINE METHYLASES
VOL. 122, 1975
TBmLE 1. Distribution of [3HJ5-MeC in pyrimidine tracts isolated from in vivo labeled A * B mec- (N-3) DNA and X-K-12 mec+ DNA Total ['HJMeCa (%) for phage: A K mec+ A* imec- (N-3)
Pyrimidine isostich no.
1
0
0
2
6.6
5.0
Compositional isomer
C2
CT 65.9
3
13.2
4
64.8
16.2
7.0
A K mec+
6.2 0.4
4.9 0.1
CS
3.0
CT2
62.4 0.5
7.4 57.4 0
C4 CST
0 3.4 9.8
1.1 5.4 9.7
C3T2 C2T3
0.1 1.1 5.8
0.1 0.9 4.5
C4T
5.5
A*B mec- (N-3)
C2T
C2T2 5
Total [3H]MeCa
6
1.6
1.8
NA
NA
NA
7
5.6
4.5
NA
NA
NA
>7
8
20
400
15-
(N~~~~~~~~~~~~~~~~
OFRACTION NUMER
FIG. 3. Chromatography of pyrimidine tracts from depurinated 3H-fd-mec- (N-3) DNA. Depurinated, 3H-labeled fd mec- (N-3) DNA (- 30,000 counts/min) and depurinated carrier calf thymus DNA (75 mg) were co-chromatographed on DEAE celMlose in 7 M urea-0.1 M sodium acetate, pH 5.5. The column was washed extensively with 7 M urea-0.1 M sodium acetate (pH 5.5) and then eluted with a linear gradient of 0 to 0.25 M NaCI in 7 M urea-0.1 M sodium acetate (pH 5.5) until pyrimidine tract no. 8 was eluted. The column was then washed with 1 M NaCI in 7 M urea-0.1 M acetate (pH 5.5). Fraction numbers 1 to 100 were 5.2 ml each; the gradient fractions were 2.6 ml each. The graphs are plotted as in Fig. 1. -
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134
plasmid methylase. For this purpose, calf thymus DNA was methylated with a crude extract derived from B mec (N-3) cells in the presence of S-adenosyl-L- [methyl-3H ]-methionine, and the DNA was subjected to depurination and pyrimidine tract analysis (see Materials and Methods). The distribution of MeC among the various pyrimidine isostichs and their compositional isomers is summarized in Table 3. The MeC distribution in calf thymus DNA pyrimidine tracts was similar to that for X DNA (see Table 1), although some differences in the relative labeling of' isostichs 2 and 3 were observed. Methylation of X DNA in vitro: saturation levels of methylated bases. The data presented above reveal a striking similarity in the distribution of MeC in various pyrimidine tracts. These results were unexpected, especially in view of the twofold increase in MeC -1000 content for X or fd DNA propagated in mecF.CZ (N-3) versus mec+ hosts. If the additional MeC lzz.l produced by N-3 were in a new sequence(s), we c might have anticipated a change in the MeC 4I-i :50 distribution among pyrimidine tracts. However, -500 if on the other hand, the MeC levels that are produced on X and f'd DNAs reflect a difference in the in vivo rate of methylation or availability ";:k of' sites prior to phage DNA maturation, then we should expect that A DNA would be methylated in vitro to the same extent by the K12 mec+ and N-3 methylases. This possibility was tested by 100 20 80 0 40 60 using cell-free extracts derived from K12 mec+ FRACTION NUMBER FIG. 4. Rechromatography of 3H-labeled isostich and K12 mec- (N-3) cultures (see Materials and no. 3 from fd.mec- (N-3) DNA. The 3H-containing Methods). The results summarized in Table 4 demonisostich (Fig. 3) was rechromatographed on a column (25 by 0.5 cm) of DEAE cellulose in 7 M urea-0.1 M strate that the mec+ extract is capable of formic acid (pH 3.5), as in Fig. 2. Fractions were 2.6 methylating various A DNAs in vitro to an ml each. extent similar to that observed with the mec-
isomer (Fig 4). This result is the same for fd-mec+, fd-mec+ (N-3), and fd-mec- (N-3) phage DNA (Table 2). We should point out that C2T is also the major MeC-containing isomer in A DNA. Analysis of MeC-containing pyrimidine tracts from calf thymus DNA methylated in vitro by a crude extract of E. coli B mec(N-3) cells. Calf thymus DNA has a base composition (44% GC) significantly different than A DNA (50% GC); in addition, due to the large amount of thymus DNA per unit genome, it contains a greater possible variety of nucleotide sequences than A DNA. Thus, we were interested to see how these factors affect the distribution of MeC residues produced by the
TABLE 2. Distribution of [3H]5-MeC in pyrimidine tracts isolated from in viuo labeled fd mec- (N-3), fd. mec+ and fd mec+ (N-3) DNA
Pyrimidine fd DNA
isostich no.
fd
mec-
(N-3)
3
3Total THoMeCa ~~(%)
100
.Total
Copstoa
Compositional ismr C3 C 2T
CT2 fd mec+
fd.mec- (N-3)
3
3
100
100
[3H]MeC % 8
92 0
C3
1
C2T CT2
99 0
C33
15 85
C2T
a The data for fd mec- (N-3) DNA are taken from Figs. 3 and 4. The data for fd mec+ and fd mec+ (N-3) were obtained from separate experiments.
VOL. 122, 1975
DNA-CYTOSINE METHYLASES
(N-3) extract. It is interesting to note that A * K mec+ DNA, which already contained approximately 65 MeC per DNA, is methylated to similar extents by both mec+ and mec- (N-3) extracts. Thus, in vitro the mec+ and N-3 methylases appear to recognize the same sites on X DNA. One unexplained discrepancy in the data must be mentioned; namely, the calculated number of MeC per X DNA is about one-half that observed for in vivo labeled XA mec- (N-3) DNA (extracted from mature virus particles). We know that the data summarized in Table 4 were obtained from DNA methylated to saturation. This was verified independently in the following manner. When comparable assay mixtures were prepared and "4C-methyl label incorporation attained its plateau value, addition of either mec+ or mec(N-3) extract had no effect on this value, but addition of more DNA led to resumption of incorporation up to a level directly proportional to the amount of added DNA. Therefore, the observed extents of methylation are not artifacts of enzyme inhibition (inactivation) during the in vitro incubation. DISCUSSION In this communication we have analyzed the distribution of [3H]5-methylcytosine among pyrimidine tracts for phage propagated on various host cells. The distribution of MeC among pyrimidine tracts was found to be the same for phage DNA labeled in E. coli K12 mec+ versus E. coli K12 mec- (N-3) bacteria. These results are surprising in view of the twofold higher MeC content for phage grown in N-3 containing cells (17). However, two alternative models can account for the results. In the first model, the N-3 enzyme recognizes the K12 mec+ sequence(s) and a new related sequence(s) differing from the former either in the nature of the purine residues outside the pyrimidine runs, or in the pyrimidine sequence within the runs. This notion can be illustrated for the sole MeC-containing tract in fd DNA, namely, C2T. This tract must originate from a site on the DNA containing the sequence The difference NpPu (pCpCpTp)PupN between the K12 mec+ and the N-3 methylation recognition could reside at the proximal purine (Pu) residues (or distal bases N etc.) or the difference might lie within the tripyrimidine ...
...
135
TABLE 3. Distribution of [3H]5-MeC in pyrimidine tracts isolated from in vitro labeled calf thymus DNA Pyrimidine isostich no.
Total 3H-MeCa (%)
Compositional isomer
Total sH-MeCa (%)
1
7
3.1
NA
NA
The data were obtained from experiments analogous to those for A and fd DNA. NA, not analyzed. a
methylation in mec+ versus mec- (N-3) host cells. The lower phage MeC content after growth in mec+ cells may be due to differences in the mec+ versus (N-3) enzymes, or it could reflect a lower methylase concentration in mec+ cells. Several independent observations support the second model. Firstly, nothwithstanding a twofold difference in MeC content, we observed identical distributions of [3H]MeC among pyrimidine isostichs for phage grown in mec(N-3) and mec+ cells (Tables 1 and 2). Recent experiments also show that the methylated tripyrimidine sequence is C-MeC-T for both fd-mec+ and fd-mec- (N-3) DNAs (May and Hattman, manuscript in preparation). Secondly, the in vitro methylation experiments reveal that under saturation conditions the mec- (N-3) and mec+ extracts methylate cyto-
sine residues on A DNA to the same extent (Table 4). Although the MeC level produced in vitro is somewhat lower than expected, it is clear that the two methylases modify the same sequence. The second model proposes that the N-3 and number of sites. This situation is consistent K12 mec+ enzymes recognize identical se- with the second model only. Finally, only a small difference in MeC content (< 20%) was quences but that the difference in MeC levels on phage DNAs is due to a lower in vivo rate of observed for host E. coli DNA isolated from
J.BACTERIOL.
MAY AND HATTMAN
136
TABLE 4. In vitro methylation of phage X DNA by crude extracts of mec+ and mec- (N-3) cellsa Calculatedd Expt. Extrt Observedc Acceptorb Calculatedd xtrac no. DNA MeC/MeAde ['4CJMec/DNA [4C]MeAde/DNA 1
XABmecXAKmec+
2
XABmecX B mec- (N-3)
4.6
82
18
81 50
mec- (N-3)
4.3 3.3 3.8
51
19 15 14
mec+ mec- (N-3) mec+ mec- (N-3)
3.5 3.9 0.06 0.03
94 99
