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Sequence specificity of the wild-type (dam+) and mutant (damh) forms of bacteriophage T2 DNA adenine methylase
J. Mol. Bid.
(1978) 119, 361-376
Sequence Specificity of the Wild-type (dam+) and Mutant (damb) Forms of Bacteriophage T2 DNA Adenine Methylase STANLEY
HATTMAN
University of Rochester, Department of Biology Rochester, N.Y. 14627, U.S.A. HANS VAN ORMONDT
AND ADRIAN
DE WAARD
Lab. voor Fysiologische Scheikunde Sylvius Laboratoria der Rijksunivwsiteit Postbus 722, Leiden-2405, The Netherlands (Received 21 July 1977) Non-glucosylated, non-methylated phage T2 DNA was methylated in vitro with partially purified wild-type (dam + ) or mutant (darn”) T.2 DNA adenine methylase. The radioactively labeled methyladenine-containing DNA was enzymatically degraded and the resulting oligonucleotides were separated according to chain length by DEAE-cellulose chromatography. Following “fingerprinting” by twodimensional electrophoresis, we determined the sequence for various di-, tri- and tetranucleotides containing radioactive Na-methyldeoxyadenosine. From this analysis we conclude that both T2 dam+ and T2 damh contain the sequence 5’ . . . G-mA -Py . . .3’.
1. Introduction The phenomenon of host-induced modification/restriction in prokaryotes is mediated by two DNA sequence-specific activities; namely, a DNA methylase and a DNA endonuclease. Not all DNA-methylating enzymes necessarily function to protect DNA against site-specific nuclease(s). For example, bacteriophages T2 and T4 (but not related phage T6) induce synthesis of an adenine-specific DNA methylasc (S-adenosyl-n-methionine : DNA (6- aminopurine) methyl transferase (EC2.1.1 .1.37)) (Gold et al., 1964; Fujimoto et al., 1965; Hausmann & Gold, 1966) though only a small fraction of the adenine residues are methylated to produce NE-methyldeoxyadenosine (Dunn & Smith, 1958; Hattman, 1970). The fact that the phage T6 genome does not contain any mAt (Gefter et al., 1966; Hattman, 1970) would appear to rule out any fundamental genetic or physiological role for DNA methylation, although specific methylation and subsequent demethylation still cannot be ruled out as playing roles in these processes. No specific biological function has yet been established for the T2 or T4 DNA methylase. In this regard, however, starting with non-glucosylating (gt-) strains of phage T2 or T4 (Revel et al., 1965) it was possible to obtain mutants which produce an altered DNA methylase (see Revel t Luria, 1970). These t Abbreviations used: mA, NG-methyldeonyadenosine; labeled with 3H or l*C.
mA*,
N6-methyldeoxyadenosine
when
361 OOSZ-2836/7s/ll93-6176
13
$02.00/0
0 1078 Academic
Press Inc. (London)
Ltd.
362
9. HATTMAN,
H.
VAN
ORMONUT
AND
a.
DE
WAARI)
mutants, now designated damh (Brooks & Hattman, 1973), have a two- to threefold higher mA content than the parental dam.+ phage (Hattman, 1970). As a result, T2 gt - damh phage (but not T2 gt - dum + ) are resistant t,o restriction by cells lysogenic for prophage Pl (Hattman, 1970). Further studies showed that the dam” enzyme is also capable of methylating more sites in vitro than the dam+ enzyme (Revel $ Hattman, 1971; Hehlmann & Hattman, 1972). In addition to hypermethylating &hydroxymethylcytosine-containing DNA, the damh enzyme can hypermethylate certain C-containing DNAs (unpublished observations). These results could bc explained if the damh methylase has a lower specificity than the dam+ enzyme ; i .fl. the damh methylase might recognize a (shorter) sequence of bases that is contained within the dam+ recognition site. Thus, the damh enzyme would methylate all the dam+ sites as well as those sites where the 5’ and/or 3’ terminal nucleotide residues In view of these findings, it was of iuterest to preclude or reduce dam + methylation. establish the nucleotide sequence(s) met,h,ylat,ed by the T2 dam+ and ‘IT2 damh enzymes. The results of these studies are the subject of the present paper.
2. Materials and Methods (a) En,zymes Es&erickia coli alkaline phosphatasc (Worthiugton Biochemical Corp.) was stored at -20°C at a concentration of 1 mg/ml in 20 mrv-Tris.HCl (pH 8.0), 10 mM-MgCl,; prior to freezing, the enzyme solution was heated at ‘70°C for 10 min to reduce any contaminating phosphodiesterase activity. Crotulus adamantezbs venom phosphodiesterase (Worthington Biochemical Corp.) was stored at -20°C at a concentration of 1 mg/ml in 0.1 M-Tris .HCl (pH 7.5); prior to freezing, the enzyme was treated by the procedure of Sulkowski & Laskowski (1971) to reduce any contaminating 5’-nucleotidase activity. Venom phosphodiesterase (Boehringer-Mannheim) was stored at 4°C as shipped from the maniafacturer; no significant 5’-nucleotidase activity was detected in this preparation. Calf spleen phosphodiesterase (Boehringer-Mannheim) was stored at 4°C as shipped from the manufacturer. Pancreatic DNase I (electrophoretically pure, Worthington Biochemical Corp.) solutions were prepared immediately before use. E. coli exonuclease I was purified by Dr Maureen May from the ammonium sulfate fraction I (Lehman & Nussbaum, 1964), generously provided by Dr Ray Wu with a detailed description for further purification The enzyme was concentrated by precipitation by DEAE-cellulose chromatography. with (NH,)&lO,, dissolved in 1.0 ml of 50 mM-Tris.HCl (pH 7.5) and stored frozen at - 20°C. Wild-type (dam + ) and mutant (damh) forms of phage T2 DNA adenine methylase were purified by MS Joan Brooks and Dr Malthi Masurekar, respectively. (b) Isotopes and analytical
materials
&‘-adenosyl-L-[methyl-3H]methionine and S-adenosyl-L-[meth$-14C]methionine were obtained from Amersham/Searle. The 5’-deoxydinucleotides pApC, pApT, pApA and pApG were obtained from Collaborative Research, Inc., Waltham, Mass. 2’-Deoxyadenosine was purchased from Calbiochem. DEAE-cellulose paper (DE81) was purchased from Whatman, Inc. Autoradiographs were made on Kodak RR/R54 Royal X-O Mat or medical X-ray film; development was with Kodak D19. Acid fuchsin and methyl orange were purchased from Fisher Scientific; xylene cyan01 FF was from Eastman Organic Chemicals.
(c) Digestion. with pancreatic DNaee Non-glucosylated, non-methylated phage T2 gt- damh dam-l DNA (Brooks & Hattman, Radioactively labeled 1973) was used as the substrate for the dam + or damh methylase. DNA was prepared by in. vitro methylation using S-ndenosyl-L-[methyZ-3H]methionine or ,S-adenosyl-n-[~&hyZ-14C]mothionine as the methyl donor (see Table 1). The labeled extensively against water DNA was deproteinized by extraction w*itb pl~cnol, clialyzed
TZ DNA
METHYLASE
363
SPECIFICITY
and concentrated under a stream of air. The DNA was then hydrolyzed with (elect,rophoretically pure) pancreatic DNase I by one of two procedures. (1) The DNA solution was evaporated to dryness and then suspended in 50 ~1 of a 1.0 mg/ml solution of DNase I (in 10 mM-Tris.HCl, pH 7.6 plus 10 mM-Mgcl,). After incubation at 37’C for 18 h, an additional 25 ~1 of enzyme was added. Following 3 to 4 h further incubation, the hydrolyzate was either lyophilized or evaporated to dryness under a stream of air. DNA digested by this method was subjected to S-dimensional electrophoresis (Sanger & Brownlee, 1967). (2) The concentrated DNA was digested at 37°C for 18 h in a reaction mixture (5 ml) containing 20 mM-Tris *HCl (pH 7.6), 10 mM-MgCl, and 250 pg DNase I/ml; an additional 100 pg DNase was added/ml and incubation was continued for 3 to 4 h. The reaction mixture was either frozen at - 20°C or treated with E. toll: exonuclease I (see below-). (d) Exonuclease
I digestion
as described above (method 2), glycine buffer (pH 9.2) Following DNase I digestion was added to a final concentration of 0.1 M. Then 20 ~1 of E. coli exonuclease I was added and the mixture was incubated for 4 11 at 37°C. The digest was neutralized with HCI and then frozen at - 20°C until ready for use. (e) .B’ractionation
of oligonucleotides
accorchg
to chain
length
Frozen DNA digests were thawed and diluted lo-fold with distilled water. The sample was applied to a 1 cm x 11 cm column of DEAE-cellulose (in water) ; the column was washed with 3 bed vol. of 2.5 mM-sodium acetate (pH 4.8) in 7 M-urea; fractionation of oligonucleotides on the basis of chain length was achieved by elution with a 200-ml linear acetate (pH 4.8) and 7 M-urea (Tomlinson NaCl gradient (0.0 M to 0.20 M) in 2.5 mM-sodium $ Tener, 1963). Fractions (2 ml) were collected and portions were taken for radioactivity determination. The di-, tri- and tetranucleotide peak fractions were pooled and desalted (see below). Oligonucleotides of chain length 6 or longer were not eluted under these conditions. (f) Desalting of oligonucleotides Pooled oligonucleotide fractions of a given chain length were diluted 5 to lo-fold in distilled water. The sample was applied to a l-ml bed volume column of DEAE-cellulose (in water). Urea and salts were removed by washing the column with 20 ml water and then 20 ml of 0.02 M-triethylammonium bicarbonate (pH 8.5). The nucleotides were eluted with 2 ivf-triethylammonium bicarbonate (pH 8.5); the sample was concentrated under a stream of air, and then evaporated to dryness in vacua. The dried samples were twice resuspended in water and dried in vacua on Parafilm (American Can Co.) sheets. (g) Dephosphorylation
of oligonucleotides
with alkaline
phosphatase
Nuoleotides were eluted from DEAE-paper with 2 nr-triethylammonium bicarbonate (pH 9.5) and dried on Parafilm sheets in vacua; the samples were taken up in 10 ~1 of a 0.1 mg/ml solution of E. coli alkaline phosphatase (in 50 mM-TriseHCl, pH 8.9, plus 10 mM-Mgcl,) and incubated in drawn capillary pipets for 2 to 3 h at 37%. The reaction was terminated by spotting on DEAE-paper. (h) Digestion
with snake venom
phosphodiesterme
For complete digestion, dephosphorylated oligonucleotides were suspended in 10 ~1 of a 0.1 mg/ml solution of snake venom phosphodiesterase (in 50 m&r-TriseHCl, pH 8.9, plus 10 mM-MgCl,) ; incubation was carried out as described above (section (8)). Separate experiments showed that the enzyme preparations had no detectable 5’-nucleotidase activity. For partial digestion, the enzyme concentration was 25 pg/ml and the reactions were carried out for 0, 5, 10 and 30 rnirr at 22°C. (i) Digestion with spleen phosphodiesterase Just prior to use, spleen phosphodiesterase was diluted in, and briefly 0.05 M-ammonium succinate (pH 6.5), 1 mnr-Na,EDTA and 0.05yo (w/v)
dialyzed against,, Tween 80 (Fisher
364
8.
HATTMAN,
H.
VAN
ORMONDT
AND
A.
DE
WAARD
Scientific). For complete digestion, oligonucleotides were resuspended in 10 ~1 of spleen phosphodiesterasc (0.1 mg/ml), 2’-deoxyadenosine (1.0 mg/ml) and incubated for 4 h at 37°C. For partial digestions, the enzyme concentration was 10 &ml, and reactions were carried out as described in section (h), above. Since many commercial preparations of spleen phosphodiesterase are contaminated with an enzyme that deaminates deoxyadenosine, or demethylaminates P-mcthylet al., 1974a), carrier deoxyadenosine was added t,o protect deoxyadenosine (Dugaiczyk against loss of any labeled methylated nucleosides generated by phosphodiesterase cleavage. Our preparation of spleen enzyme, however, did not contain significant de-
aminating
activity.
3. Results (a) Fmctionation
of obigonucleotides
according to chain length
As a prelude to analyzing DNA oligonucleotides containing mA, it was desirable to fractionate enzymatic digests according to chain length (isostichs). One convenient method involves ion-exchange chromatography on DEAE-cellulose in the presence (Tomlinson & Tener, 1963). Figure 1 shows the chromatographic separaof 7 M-Urea tion of oligonucleotides produced following pancreatic DNase digestion of 32Plabeled (non-glucosylated) T2 gt- DNA. Pancreatic DNase produces relatively few mononucleotides (Sinsheimer, 1954); using a 0 M to 0.25 M-NaCl gradient, isostichs of up to chain length 7 could easily be discerned (Fig. l(a)). When the DNase digest
300 L
1
Co”+rol’
X
600
25
75
50
loo
25 Fraction
50
75 (b)
(0)
FIG. 1. Separation cellulose
digested cellulose
100
number
of 32P-labeled oligonucleotides of similar chain length (isostichs) by DEAE32P-labeled T2 gt- dam” darn-1 DNA was isolated and chromatography (in 7 M-urea). with pancreatic DNase (see Materials and Methods). The digest was subjected to DEAEchromatography (in 7 M-Urea) before and after further digestion with E. coli exouuclease 1
((a) and (b), respectively). The conditions and Methods section. The numbers indicate
for chromatography the chain
lengths
are described of the various
in the Materials
isostichs.
T2
DNA
METHYLASE
365
SPECIFICITY
was treated with E. co& exonuclease I prior to column chromatography, most of the 32P label was recovered in mononucleotides and dinucleotides (Fig. l(b)) ; some 32P label was still observed in the trinucleotide region indicating that a limit digest was not achieved. non-methylated) was used as the subT2 gt - darn” dam-l DNA (non-glucosylated, strate for in vitro methylation by the T2 dam+ and damh enzymes, respectively ; separate experiments verified that mA was the only labeled base produced. Figure 2 shows the chromatographic fractionation of [3H]oligonucleotides produced following successive treatment with pancreatic DNase and exonuclease I. The [3H]dinucleotide peak fractions were pooled, desalted and analyzed further (see below).
damh
dam+ I
I
2250
t = 2 2000 lo k za .z 1000 Lo Tl x. F t 750 8 a e ,I 500 -
3
3
I
250 L2 25
50
75
loo
25 Fraction
?( z 59
75
loo
number
(0)
(b)
FIG.
2. Separation of [3H]methyl-labeled isostichs by DEAE-cellulose chromatography. T2 gt- damh dam-l DNA was methylated in vitro by either (a) T2 dam+ or (b) T2 dam” methylase, digested successively with pancreatic DNase and E. co& exonuclease I, and subjected to DEAEcellulose chromatography (see Materials and Methods).
(b) Characterization
of N6-methyldeoxyaden~osine-containing
dinucleotides
The [3H]mA-containing dinucleotides were analyzed by high voltage electrophoresis on Whatman 3MM paper; four authentic deoxydinucleotide markers containing 5’-A were co-electrophoresed in the same track as the sample. At pH 1.9, three peaks of 3H radioactivity were observed (Fig. 3(a)). Two negatively charged [3H]dinucleotides co-electrophoresed with marker pApT and slightly ahead of marker pApG, respectively. The third [3H]dinucleotide migrated toward the cathode with
366
S. HATTMAN,
H.
VAN
ORMONDT
AND
il.
DE
WAARD
pApA and pApC (not resolved at pH i-9). However, at pH 3.5, all four marker dinucleotides were separated (Fig. 3(b)); it is clear that 3H label is in the pApC region and not in pApA. The 3H-labeled (MA*, G) and 3H-labeled (mA*, T) were not resolved under these conditions; the parentheses indicate that the sequence is not known. Similar results were obtained with the dinucleotides from both dam+ and dumb methylated DNA. The absence of 3H-labeled (mA*, A) is in excellent agreement with the observations of Vanyushin et al. (1971) who studied ma-containing purine tracts in T2 DNA; they found mA in the purine dinucleotide (mA, G), and not in (mA, A) nor (mA, mA). Complete digestion with spleen or snake venom phosphodiesterase showed that the sequences of the three dinucleotidesare G-mA*,mA *-hmC, and mA*-T (nucleotide sequences will be written from left to right to correspond to the 5’ to 3’ polarity). This suggests that the dam+ and damh methylases recognize sequences containing
(0)
’ pti 1.9
I
I
I
I
I
500 400
PAPG -
-
-
300
PAPT
-
200 100
-5
< 2 u ,: Ii x 2
b)
I
0
5
I
I
PAPC -
350
IO
I5
-20
I
I
I
PAPA -
-r
PAPG PAPT --
pH 3.5 300
I? 250 200 150 I00 50
5
IO 15 20 25 Distance from orgIn km1
I30
35
FIG. 3. Electrophoretic analysis of [3H]mcthyl-labeled dinucleotides from T2 gt- dam” dam-l dinucleotide fractions (see Fig. 2) DNA methyl&ted in vitro with T2 dam “. The [3H]methyl-labeled were pooled, desalted and subjected to paper electrophoresis (Whatman 3MM) at either (a) pH l-9 or (b) 3.6. Authentic deoxynucleotides of known sequence were included ES ultraviolet absorbs+nce markers. After the paper (2 cm x 67 cm) w&s dried, l-cm strips were cut and placed in scintillation vials; 0.5 ml water plus 6 ml scintillation fluid were added, and the 3H radioactivity W&B measured in a Packard scintillation counter.
T2
DNA
METHYLASE
367
SPECIFICITY
G-mA*-hmC and G-mA*-‘I’. We should point out that the ratio of 3H label in G-mA*/(mA*-T + mA*-hmC) was greater than unity; this is probably due to the fact that pancreatic DNase shows some cleavage specificity. It generally produces 5’-hmC rather than 3’-hmC-containing dinucleotides; i.e. there is a preponderance of 5’-hmC-N weraus 5’-N-hmC (van Ormondt & Hattman. 1976). This has also been observed with pancreatic DNase digests of C-containing DNA (Dugaiczyk et aZ., 19746; our unpublished observation). Thus, the recovery of mA*-hmC, but not hmC-mA*, indicates that the latter sequence is probably not present. (c) Two-dimensional
fingerprints
of [14C]methyl-labeled
oligonucleotides
In order to obtain more detailed information about the methylation sequence(s), it was necessary to examine larger ma-containing oligonucleotides. Previous studies from our laboratory established the two-dimensional fingerprint for hmC-containing, non-glucosylated T2 DNA (van Ormondt & Hattman, 1976). After in vitro methylation and pancreatic DNase digestion, the [14C]methyl-labeled oligonucleotides were ta,ken either directly for fingerprinting, or the di-, tri-, and tetranucleotides were first purified by DEAE-cellulose chromatography (using a 0 M to 0.20 M-NaCl gradient)
I
dam+
25
50
75
25
loo Fmction
(0 1
50
75
100
number (b)
FIG. 4. Separation of [14C]methyl-labeled isostichs by DEAE-cellulose chromatography. T2 gt- duw@ danz-1 bNA was methylated in vitro by either (a) T2 dam + or (b) T2 dam” methylsses, digested with pancreatic DNase, and subjected to DEAE-cellulose chromatography. The di-, tri-, and tetranucleotide peak fractions (indicated by braces in the Figure) were pooled, desalted and analyzed further (see text).
368
S. HATTMAN,
H.
VAN
ORMONDT
AND
A. DE
WAARD
a,nd then fingerprinted The combination of these two methods enabled us to assess more easily the nature of the various oligonucleotides. Figure 4 shows the chromatographic separation of [14C]methyl-labeled oligonucleotides produced by pancreatic DNase digestion of in vitro methylated T2 gt- damh dam-1 DN.4. The tri- and tetranucleotide regions were collected, desalted and subjected to two-dimensional electrophoresis. Fingerprints of the purified tri- and tetranucleotide fractions are shown in Figures 5 and 6, respectively. The nucleotides were eluted from the DEAE-paper, had their 5’-terminal phosphate groups removed by treatment with E. coli alkaline phosphatase, and were then fractionated further by ionophoresis on DEAE 81-paper at pH 3.5 ; in most instances, another ionophoretic fractionation was carried out at pH 3-5 and/or pH l-9. This procedure allowed us to sepa.rate a variety of sequence isomers and compositionally related oligonucleotides that are barely resolved in the two-dimensional fingerprint (Sanger & Brownlee. 1967). The sequence of each nucleotide (mixture) was then determined by partial hydrolysis with spleen and snake venom phosphodiesterases (see below).
(d) Sequence asalysis
of labeled N6-methyldeoxyadenosine-containing
tGnuc1eotide.s
Table 1 contains a summary of the sequences deduced for the mA*-containing trinucleotides (Fig. 5). The base composition of each nucleotide could be inferred, in part, from its relative position in the two-dimensional fingerprint, and the R, values? (following dephosphorylation) on DEAE-paper at pH 3.5 and 1.9 (knowledge of the mA*-dinucleotides was also a major aid in this analysis). The sequence of nucleotide 1 must be mA*-hmC-hmC because, in addition to bhe above criteria, we know that mA*-hmC and not hmC-mA* is observed among the mA* dinucleotides (see section (b), above). It is important to note here that, under the conditions of venom exonuclease digestion, no partial degradation products were seen. It was subsequently observed that all oligonucleotides containing a 3’-terminal hmC residue were relatively resistant to venom exonuclease digestion. This property proved to be a useful diagnostic tool, as will be discussed below (for nucleotides 3 and 4). Nucleotide 2 is a minor species that is barely visible in Figure 5. Based on the criteria outlined above. we believe the only sequence possible for it is mA*-hmC-A. Nucleotide 3 appears to be a mixture of mA*-T-hmC and mA*-hmC-T; the absence of mA*-T as a partial digestion product is attributed to the presence of the 3’-terminal hmC residue. Nucleotide 4 appears to be primarily hmC-G-mA* ; however, since venom exonuclease digestion never completely degraded the starting material, we believe that the G-mA*-hmC sequence isomer is present. This would be consistent with the observed dinucleotides, G-mA* and mA*-hmC (and with the observed tetranucleotides, G-mA*-hmC-hmC and G-mA*-hmC-T (see section (e), below). Included in nucleotide 4 was another minor species which was not analyzed: it is likely that this is the mA*-hmC-G isomer. Trinucleotides 6 through 8 could be established with little ambiguity. Nucleotide 9 is present as a minor component (note: appears to be mainly T-G-mA * ; G-mA*-T G-mA*-T was expected to be a major species, since G-mA* and mA*-T are major dinucleotides). mA*-T-G is also a minor component of spot 9. We concluded that t IZB value is the distance
migrated
by oligonuclcutide/distancc
migrated
by blue dye marker.
60
70
(b)
03
04
<:35
f
8.D.
-+E-
FIG. 5. (a) Radioautograph of a 2-dimensional ionophoretic separation of trinucleotides purified from a pancreatic DPl‘ase digest of phage Td gt- dum” and [14C]mothyi-labeled S-adenosyl-I,-mcthionine. dam-1 (non-glucosylated, non-methyleted) DNA that had been methyl&& with the T2 dam + methyl&se In the first dimension, ionophoresis was on cellulose acetate at pH 3.5 (pyridine/acetato buffer); aftor transfer to DEAE 81.paper, ionophoresis in the second dimension was at pH 1.9. The hatched zone indicates the position of the blue dye, xylene cyan01 FF (spot,ted on both sides of the origin on cellulose acetate). Film exposure was for 9 days. (b) Diagram of the radioautograph. The numbered spots correspond to the oligonucleotide numbers given in Table 1. Broken lines indicate minor spots; thin lines indicate intermediat.e spots and thick lines indicate major spots. P.1). and B.D. refer to the pink dye (acid fuchsin) and blue dye, respectively.
IO 98
(0)
(b)
FIG. 6. (a) Radioautograph of a 2-dimensional ionophoretic separation of totranucleotides purified from a pancreatic DNase digest of phage T2 gt- damh dam-l DNA that had been methylated with the T2 darn” enzyme (see the legend to Fig. 5). Film exposure was for 7 days. (b) Diagram of the radioautograph.
1.9
1.31 1.35 2.45 2.41 2.00 N.D. N.D. 2.35 1.96 1.52 1.65 1.76
RB values$ 3.5 pH
1.40 1.35 1.45 1.39 1.20 1.10 0.47 1.15 1.00 0.90 1.10 1.20
pH
trilsucleotides
gt-
mA*
dig&ion products Spleen
phage T2
N.D. N.D. mA*p mA*p mA* N.D. N.D. mA*p Gm-A*; mA* mA*; G-mA* mA*p; G-mA*; mA*p
Partial
from with
damh
DNA
None N.D. None mA*-hmC pmA* N.D. N.D. mA*-T; mA* pmA* pmA* pmA*; G-mA*; mA*-T; mA*
phosphodiesterase Venom
dam-l
1
mA*-T
from:
§ trinucieot.ide
sequence?
methylase
mA*-hmC-hmC mA*-hmC-A mA*-T-hmC mA*-hmC-T hmC-G-mA*; (G-mA*-hmC) (mA*-hmC-G) (?) Tetranucleotide mA*-T-A A-G-mA* G-G-mA* T-G-mA*; (G.mA*-T; mA*-T-G) mA*-T-T
Deduced
modi$ed with T2 DNA
Non-glucosylated, non-methylated T2 DNA (from T2 gt- dam” dam-l) was methylated in vitro with purified preparations of DNA adenine methylase induced by wild-type T2 phage (dam +) or mutant (damh) using [14C]methyl-labeled S-adenosyl-n-methionine as the methyl donor. 3000 units of T2 dam+ enzyme were added to a standard reaction mixture (Brooks et al., manuscript in preparation) containing 250 pg of substrate DNA. After 60 min of incubation at 37”C, an additional 1500 units of enzyme were added; after a further 60.min incubation period, the DNA was extracted and dialyzed against water. A similar reaction was carried out in parallel, with 1500 units of T2 dam” methylase and 200 pg of DNA (300 units of dam” enzyme was added after the first 60-min incubation period). The stoichiomet)ry of methylation at saturation was calculated to be 3000 and 1500 methyl groups added per phage DNA by urn” and dam + , respectively. t The nucleotidc numbers are t.hose shown in Fig. 5(b). The 2.dimensional trinucleotide fingerprints for damh and dctm + were identical. 1 Following dephosphorylation with alkaline phosphatase, relative ionophoretic mobilities (RB) were measured with respect to the blue dye, xylene cyan01 FF, by eleotrophoresis on DEAE 81.paper at pH 1.9 and pH 3.5; thedye was allowed to migrate at least 12 cm from the origin. The figures listed are average values; generally, the variation was -C 10% of the mean value and, in a given experiment, t,he 11, values tended to be uniformly higher or lower than the mean value. N.D., not done. 5 The partial digestion products were identified from their ionophoretic mobilities on DEAE 81.paper at pH 3.5 and pH 1.9. These were also compared of 0.4 to 0.5). Since the dinuoleoside monoto values for a2P-labeled oligonucleotides which lacked mA (at pH 3.5, the deoxynucleoside, mA *, has a mobility phosphate, G-mA* , and the mononucleotide, pmA* or mA*p, have similar mobilities at pH 3.5, it was necessary to analyze these further. (The 3’, 5’ phosphodiester bonds will generally be represented by dashes ; terminal 3’ or 5’-phosphates will be indicated in the appropriat,e positions.) This was carried out to flatby one of two methods. (1) Aftes autoradiographic analysis and location on DEAE-paper, the spots (Es = 1.8 to 1.9) were eluted and subjected plate electrophoresis on (2.5 cm x 57 cm) Whatman 3MM paper (0.1 M Tris, pH 8.0) to separate the mononucleotide and dinucleoside monophosphate. Authentic markers were co-electrophoresed and located by their ultraviolet light absorbance; l-cm width strips were cut and the r*C radioactivity counted. (2) DEAE-paper electrophoresis at pH 1.9 also proved to be a convenient method, since, at this pH, G-mA* migrates much slower than pmA* or mA*p. N.D., not done. 11 The minor species observed are enclosed in parentheses. See text for discussion.
1 2 3a 3b 4a 4b 5 6 7 8 9 10
Sucleotidet number
Methylated
TABLE
mA*.‘F.‘r.
mA*p;
G-G.mA*;
0.68
0.7’1
0.35
10b
1oc
118
G-m&4*;
mA*-T-hmC mA*
analyzed
pmA* mA*-(T, hmC) pmA*; G-mA*; hmC-G-mA* mA*-T; pmA*;
Not
A-G-mA*; G-mA*; mA* mA*p mA*; mA*p; m.4*-(T, hmC) (T-G-mA*/G-m.4*-T) mA*p; hmC-G-mA*/G-mA*-hmC
hmC)
0.43 047 0.59
mA*-(T,
mA*p
modijied
pmA*
mA*-hmC-A
mA*.‘p
mA*-T-hmC
G-mA*-hmC
mA*-hmC-A mA*-T; mA*-(T,hmC) mA*-hmC or mA*-hmC-hmC t unidentified products mA*-T-hmC G-mA*-hmC
NOW
NOM!
8 9 10a
mA*p;
;
DNA
phosphodiest,erase from?: Venom
mA*p mA* -hmC-hmC
with
o.ti4 0.76 0.72 0.73
produck
5a 5b s I
digestion Spleen
damh dam-l
8.D. N.D. N.D. m.4*p
Partial
from phage T2 gt-
0.93 0.86 0.63 0.85
R, valuet at pH 3.5
tetranucleotides
1 2 3 4
Nuoleotide numberf
Methylated
TABLE 2
tetranucleotido
methylasei sequence
3
A-G-G-m-4*
mA*-T-hmC-A G-mA*-hmC-hmC (G-mA*-hmC-A/A-G-mA*-hmC) (?) mA*-hmC-A-G + dephosphorylated G-mA*-T-hmC A-A-G-mA* mA*-(T, hmC)-G or mA*-(T, hmC)-;l-P hmC-G-mA*-T; hmC-T-G-mA*; G-mA*-hmC-T mA*-T-hmC-(T or G) T-G-mA*-hmC/T-hmC-G-mA* G-mA*-T-hmC; mA*-1’.T-hmC
mA*-hmC-hmC-hmC mA*-hmC-A-hmC m-4*-hmC-A-A mA*-(T, hmC)-hmC’ mA*-hmC-T
Deduced
with T2 DNA
-
mA*-T-A; 4.G-m4* ‘r-G-mA*/G-mA*-T; mA*p; mA* T-G-mA*/G-mA*-T; A-G-mA*; G-mA* ; mA*-T; mA*p; mA* G-G-mA*; T-G-mA* G-G-mA*; mA*-T G-mA*-T ; mA*p mA*-T-T; G-mA* mA*-T-T; G-mA* T-G-mA*; mA*-T mA*; G-G-mA* mA*p mA*-T; mA*-T-T G-mA*; mA*-T-A; ; mA*
T-G-mA* ; pmA*
mA*-T-T; mA*-T; T-G-mA*/G-mA*-T
G-mA*-T; G-G-mA*
pmA* G-G-mA*; pmA* mA*-T; G-mA*
mA*-T-T; A-G-mA*
mA*
T-G-mA*/G-mA*-T/mA*-T-G G-mA*; pmA*; mA*-T
mA*-T-T-G; G-mA*-T-T
mA*-T-T-G; G-G-mA*-T; T-T-G-mA*;
mA*-T-A-G mA*-T-G-A
A-G-mA*-T; mA*-T-T-A’ T-A-G-mA*’ G-G-G-mA* G-G-mA*-T; mA*-T-G-(G
T-G-mA*-T
G-mA*-T-T T-G-mA*-T T-G-G-mA*
; G-T-G-mA* T-G-G-mA* or T); G-mA*-T-T
mA*-T-G-A ; G-mA*-T-A
T-A-G-mA*; A-T-G-mA*
t See the legend to Table 1. Some of the tetranucleot,ides were not detected in the fingerprint (Fig, 6); however, they were obtained from an independent preparation and are included in the Table. 1 The nucleotide numbers are those shown in Fig. 6(b). The 2.dimensional tetranucleotide fingerprints for dam” and dam+ were identical. product, from 5 The base composition was inferred from the position of the oligonucleotide in the fingerprint, from the R, value of the dephosphorylated the products of (or resistance to) partial exonuclease digestion, and from the observed mA*-containing trinucleotides (Table 1). This strategy, however, did not always lead us to an unambiguous sequence assignment. In cases where we could not distinguish between sequence isomers, we have either enclosed the relevant bases in parentheses or written both sequences, connected by a slash (c.g. nucleotide lob could contain T-G-m-4*-hmC and/or T-hmC-G-mA*). N.D.. not done.
0.48 ; 0.53
13b
0.46
0.33 0.40
12a 12b
0.43;
0.49
llc
13a
0.4”
llb
374
S. HATTMAN,
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ORMONDT
AND
A. DE
WAARI)
nucleotide 10 is mA*-T-T, and not mA*-T-G, due to its position in the fingerprint relative to G-G-mA* and T-G-mA*, and to its higher R, value (at pH 3.5) than T-G-mA*; for example, since sequence isomers with a 5)-A residue tend to have a somewhat lower R, value than their counterparts containing a 3’-A residue (Sanger & Brownlee, 1967 ; our unpublished observations), if spot 10 were mA*-T-G, it, would not have had a higher R, value than T-G-mA*. Finally, the observa,tion that G-mA*-T-T is among the mA*-tetranucleotides (see section (e). below) is consistent with the occurrence of mA*-T-T. Based on the observed dinucleotides, mA*-hmC, mA*-T, and G-mA*, we had anticipated that G-mA*-T and G-mA*-hmC would be among the major mA*trinucleotides. As shown in Table 1, these sequences represented only minor species ; mA* was observed mainly at the 5’ or 3’ position of the trinucleotides characterized. It is possible that the N6-methyl group of mA* exerts some influence on the specificity of pancreatic DNase cleavage. From the results summarized in Table 1, it appears that a variety of methylated trinucleotide sequences are produced by both dam+ and da,mh; namely X-G-mA*, G-mA*-Py, and mA*-Py-N. The only species for which there is some uncertainty is mA*-hmC-G. Nevertheless, it appears that the recognition sequence for both enzymes is simply N-G-A-Py-N: however, we do not believe that all these sites can be methylated with equal ability by dam+ and damh (see Discussion). (e) Xequence analysis
of tetranucleotides
tetranucleotides obtained Figure 6 shows a fingerprint of purified [‘“Cl methylated after pancreatic DNase digestion and DEAE-cellulose chromatography of T2 gtdamh dam-l DNA methylated by the T2 damh enzyme (see Materials and Methods). Table 2 contains a summary of the sequence analysis of the various nucleotides. As with the trinucleotides, we observed no reproducible, significant differences in the DNA ; fingerprints of tetranucleotides from T2 damh veraus T2 dam+ methylated furthermore, due to the larger number of sequence isomers in each spot, compared to trinucleotides, it was even more difficult to detect any quantitative differences among tetranucleotides. Based on the general methylation sequence, 5’. . . N-G-mA*-PyN.. .3’, derived from the trinucleotide analysis, there are 64 possible tetranucleotide sequences. We observed approximately 213 of t,hese combinations, which are likely to correspond to the most frequently occurring methylated tetranucleotide sequences in T2 DNA. The observation of tetranucleotides containing G-mA*-hmC and GmA*-T (nucleotides 5b, lOa, b, c and lib, c) confirmed these trinucleotide assignments.
4. Discussion Although the cleavage site for many site-specific endonucleases has been established, only a relatively small number of DNA methylation sequences have been elucidated. In practice, the determination of methylation sites is a more tedious procedure. This generally starts with the in vitro methylation of a substrate DNA with (partially) using either [14C]- or [3H]methyl-labeled Spurified DNA methylase activity, adenosyl-L-methionine as the methyl donor. The DNA is then enzymatically degraded and the resulting oligonucleotides are fractionated and sequenced. The methylation sequence is deduced from the overlapping sequences determined for the various site is a unique sequence of radioactive oligonucleotides ; when the methylation
T2
DNA
METHYLASE
375
SPECIFICITY
four to six residues, the analysis is simplified by the presence of only a small number of labeled nucleotides. Using this general approach we have investigated the site(s) methylated by the dam + and da+ forms of the T2 DNA adenine methylase. The damh enzyme methylates two- to threefold more sites than the dam+ enzyme; e.g. approximately 3000 and 1000 mA residues per mature phage T2 gt - DNA are produced by damh and darn, + , respectively (Hattman, 1970). (We estimated that the dam+ methylation sequence should be about four to five (specific) nucleotide residues long.) Thus, we hoped that our analysis would reveal mA-containing oligonucleotides unique for damh methylation. Contrary to this expectation, we observed the same methylated di-, tri- and The methylated sequence tetranucleotides for the dam + and damh methylases. consistent with these oligonucleotides is 5’. . . N-G-mA*-Py-N . .3’. Further support for this comes from the level of in vitro methylation attained; e.g. 3000 mA residues per T2 DNA (see Table 1) corresponds to about 300;;, of the calculated number of G-A-Py sequences (based on random distribution of bases in T2 DNA). Recent experiments have shown that it is possible Do reach higher methylation levels and approach the theoretical maximum (unpublished data). The results presented extend those reported for T2 dam+ methylation of micrococcal DNA, where the only sequence methylated was G-mA*-T-C (van Ormondt et al., 1975). Tt is not clear why we observe a lower methylation specificity with T2 gl- DNA; it is not due to the presence of hmC in T2 phage DNA, since we also find G-mA*-C in h DNA methylated by T2 dam+ (unpublished observations). Tt should also be noted that the occurrence of certain methylated tetranucleotides (e.g. G-mA*-hmC-hmC, G-mA*-T-T and G-mA*-T-A) indicates that S-fold rotational symmetry is not required for sequence recognition. This has been observed with the et al., 19740) M.EcoB modification methylase (van Ormondt et al., 1973; Dugaiczyk and the M.EcoPlf modification methylase (S. Hattman. J. Brooks & M. Masurekar: manuscript in preparation). Our analysis failed to reveal any qualitative difference(s) in methylation specificity between T2 dam+ and T2 damh; furthermore, we did not observe any significant. reproducible quantitative differences in the occurrence of mA*-oligonucleotides (as judged by visual inspection of autoradiograms). However, a failure to detect any qua’ntit’ative difference might have been the result of selecbive degradation during pancreatic DNase digestion. This is a plausible explanation for the low recovery of mA*-hmC, G-mA*-hmC and G-mA*-T; we have also observed this with methylated C-containing oligonucleotides (unpublished data). Therefore, it is difficult to assess to what extent pancreat’ic DNase cleavage specificity might have been a factor in our failure to detect qualitative or quantitative differences between T2 dam+ veTsus T2 dam,h methylation. Although both methylases produced G-mA*-Py, it is possible that t,he two enzymes do not methylate all sites with equal ability (further studies are in progress). In this sense, dam+ might have a low affinity for certain of the G--A-Py sequences; the mutation to damh might result in an altered enzyme which can methylate the “low affinity” sites more readily. A change in nucleotide sequence recognition has been observed under special conditions in other systems. It is known that ccrt’ain restriction enzymes do not cleave all sites at the same rate (Thomas $ t ~l~Ec01’1 is t,he modification methylase used to identify restriction and modification 1973).
specified by phage Pl (the enzymes is that suggested
three-letter by Smith
designation & Nathans,
876
S. HATTMAS,
H.
VAN
ORMONDT
AiWI
A. DE
WAARD
Davis, 1975) and that in vitro substrate specificity can be lowered by varying the pH and ionic conditions (Polisky et al., 1975). In the case of the T2 methylase forms, the change in sequence specificity is the result of a mutational alteration; it remains to be seen whether it will be possible to obtain similar mutants for other DNA methylases. Finally, the damh mutant was originally isolated from T2 gt - dam + as being resistant to restriction by phage Pl, The Pl-modification enzyme has been reported to produce the sequence 5’. . A-G-mA*-T-C-T.. .3’ (Brockes et al., 1974) : this sequence should be recognized by both dam + and dam”. Recent studies, however, indicate that the MS EcoPl sequence is 5’. . . A-G-mA*-C-Py . .3’ (S. Hattman, J. Brooks & M. Masurekar, manuscript in preparation) and that this site may be one that is not readily methylated by dam+ (J. Brooks, S. Hattman & M. Masurekar, manuscript in preparation). We grat,efully acknowledge the assistance of ,I. Brooks and M. Masurekar for their role in purifying the T2 methylases, the production of in vitro mrthylated DNA, and for mana critical discussions. We also acknowledge the participation of J. Garter in the early phases of the sequence analysis. This work was supported by a Public Health Service grant (no. AI-10864) and a Research Career Development award (no. AI-28022) to one of us (S. H.). REFERENCES Brockes, J. P., Brown, P. R. & Murray, K. (1974). J. Mol. Biol. 88, 437-443. Brooks, J. & Hattman, S. (1973). l’irology, 55, 285.-288. Dugaiczyk, A., Boyer, H. W. & Goodman, H. M. (1974a). Biochem. Biophys. Res. Commun. 56, 641-646. Dugaiczyk, A., Kimball, M., Linn, 8. & Goodman, H. M. (19746). Biochem. Biophys. Res. Commun. 61, 1133-1140. Dunn, D. B. & Smith, J. D. (1958). Biochem. J. 68, 625-636. Fujimoto, D., Srinivasan, P. R. & Borek, E. (1965). Biochemistry, 4, 2849--28X. Gefter, M., Hausmann, R., Gold, M. & Hurwitz, ,J. (1966). J. Biol. Chem. 241, 1995-2006. Gold, M., Hausmann, R., Maitra, U. & Hurwitz, ,I. ( 1964). I’roc. Nat. Acad. Sci., U.S.A. 52, 292-297. Hattman, S. (1970). virology, 42, 359-367. Hausmann, R. & Gold, M. (1966). J. Biol. Chem. 241, 1985-1994. Hehlmann, R. & Hattman, S. (1972). J. Mol. BioZ. 67, 351-360. Lehman, I. R. & Nussbaum, A. L. (1964). J. Biol. Chem. 239, 2628-2636. Polisky, B., Greene, P., Garfin, D. E., McCarthy, B. J., Goodman, H. M. & Boyer, H. W. (1975). Proc. Nut. Acad. Sci., U.S.A. 72, 3310.-3314. Revel, H. R. & Hattman, S. M. (1971). l’irology, 45, 484-495. Revel, H. R. & Luria, S. E. (1970). Annu. Rev. Genet. 4, 117-192. Revel, H. R., Hattman, S. & Luria, S. E. (1965). Riochem. Biophys. Res. Commun. 18, 545-550. Sanger, F. & Brownlee, G. G. (1967). In Methods in Enzymology (Grossman, L. & Moldave, K., eds), vol. 10, pp. 361-381, Academic Press, New York. Sinsheimer, R. L. (1954). J. BioZ. Chem. 208, 445-459. Smith, H. 0. & Nathans, D. (1973). J. Mol. BioZ. 81, 419-423. Sulkowski, E. & Laskowski, M. Sr. (1971). Biochim. Biophys. Acta, 240, 442-447. Thomas, M. & Davis, R. W. (1975). J. Mol. Biol. 91, 315-328. Tomlinson, R. V. & Tener, G. M. (1963). Biochemistry, 2, 697-702. van Ormondt, H. & Hattman, S. (1976). Anal. Biochem. 74, 207-213. van Ormondt, H., Lautenberger, J. A., Linn, S. & de Waard. A. (1973). FEBS Letters, 33, 177-180. van Ormondt, H., Gorter, J., Havelaar, K. J. & de Waard, A. (1975). Nucl. Acids Res. 2, 1391-1400. Vanyushin, B. F., Buryanov, Ya, I. & Beloeersky, A. N. (1971). Nature New BioZ. 230, 25-27.
(1978) 119, 361-376
Sequence Specificity of the Wild-type (dam+) and Mutant (damb) Forms of Bacteriophage T2 DNA Adenine Methylase STANLEY
HATTMAN
University of Rochester, Department of Biology Rochester, N.Y. 14627, U.S.A. HANS VAN ORMONDT
AND ADRIAN
DE WAARD
Lab. voor Fysiologische Scheikunde Sylvius Laboratoria der Rijksunivwsiteit Postbus 722, Leiden-2405, The Netherlands (Received 21 July 1977) Non-glucosylated, non-methylated phage T2 DNA was methylated in vitro with partially purified wild-type (dam + ) or mutant (darn”) T.2 DNA adenine methylase. The radioactively labeled methyladenine-containing DNA was enzymatically degraded and the resulting oligonucleotides were separated according to chain length by DEAE-cellulose chromatography. Following “fingerprinting” by twodimensional electrophoresis, we determined the sequence for various di-, tri- and tetranucleotides containing radioactive Na-methyldeoxyadenosine. From this analysis we conclude that both T2 dam+ and T2 damh contain the sequence 5’ . . . G-mA -Py . . .3’.
1. Introduction The phenomenon of host-induced modification/restriction in prokaryotes is mediated by two DNA sequence-specific activities; namely, a DNA methylase and a DNA endonuclease. Not all DNA-methylating enzymes necessarily function to protect DNA against site-specific nuclease(s). For example, bacteriophages T2 and T4 (but not related phage T6) induce synthesis of an adenine-specific DNA methylasc (S-adenosyl-n-methionine : DNA (6- aminopurine) methyl transferase (EC2.1.1 .1.37)) (Gold et al., 1964; Fujimoto et al., 1965; Hausmann & Gold, 1966) though only a small fraction of the adenine residues are methylated to produce NE-methyldeoxyadenosine (Dunn & Smith, 1958; Hattman, 1970). The fact that the phage T6 genome does not contain any mAt (Gefter et al., 1966; Hattman, 1970) would appear to rule out any fundamental genetic or physiological role for DNA methylation, although specific methylation and subsequent demethylation still cannot be ruled out as playing roles in these processes. No specific biological function has yet been established for the T2 or T4 DNA methylase. In this regard, however, starting with non-glucosylating (gt-) strains of phage T2 or T4 (Revel et al., 1965) it was possible to obtain mutants which produce an altered DNA methylase (see Revel t Luria, 1970). These t Abbreviations used: mA, NG-methyldeonyadenosine; labeled with 3H or l*C.
mA*,
N6-methyldeoxyadenosine
when
361 OOSZ-2836/7s/ll93-6176
13
$02.00/0
0 1078 Academic
Press Inc. (London)
Ltd.
362
9. HATTMAN,
H.
VAN
ORMONUT
AND
a.
DE
WAARI)
mutants, now designated damh (Brooks & Hattman, 1973), have a two- to threefold higher mA content than the parental dam.+ phage (Hattman, 1970). As a result, T2 gt - damh phage (but not T2 gt - dum + ) are resistant t,o restriction by cells lysogenic for prophage Pl (Hattman, 1970). Further studies showed that the dam” enzyme is also capable of methylating more sites in vitro than the dam+ enzyme (Revel $ Hattman, 1971; Hehlmann & Hattman, 1972). In addition to hypermethylating &hydroxymethylcytosine-containing DNA, the damh enzyme can hypermethylate certain C-containing DNAs (unpublished observations). These results could bc explained if the damh methylase has a lower specificity than the dam+ enzyme ; i .fl. the damh methylase might recognize a (shorter) sequence of bases that is contained within the dam+ recognition site. Thus, the damh enzyme would methylate all the dam+ sites as well as those sites where the 5’ and/or 3’ terminal nucleotide residues In view of these findings, it was of iuterest to preclude or reduce dam + methylation. establish the nucleotide sequence(s) met,h,ylat,ed by the T2 dam+ and ‘IT2 damh enzymes. The results of these studies are the subject of the present paper.
2. Materials and Methods (a) En,zymes Es&erickia coli alkaline phosphatasc (Worthiugton Biochemical Corp.) was stored at -20°C at a concentration of 1 mg/ml in 20 mrv-Tris.HCl (pH 8.0), 10 mM-MgCl,; prior to freezing, the enzyme solution was heated at ‘70°C for 10 min to reduce any contaminating phosphodiesterase activity. Crotulus adamantezbs venom phosphodiesterase (Worthington Biochemical Corp.) was stored at -20°C at a concentration of 1 mg/ml in 0.1 M-Tris .HCl (pH 7.5); prior to freezing, the enzyme was treated by the procedure of Sulkowski & Laskowski (1971) to reduce any contaminating 5’-nucleotidase activity. Venom phosphodiesterase (Boehringer-Mannheim) was stored at 4°C as shipped from the maniafacturer; no significant 5’-nucleotidase activity was detected in this preparation. Calf spleen phosphodiesterase (Boehringer-Mannheim) was stored at 4°C as shipped from the manufacturer. Pancreatic DNase I (electrophoretically pure, Worthington Biochemical Corp.) solutions were prepared immediately before use. E. coli exonuclease I was purified by Dr Maureen May from the ammonium sulfate fraction I (Lehman & Nussbaum, 1964), generously provided by Dr Ray Wu with a detailed description for further purification The enzyme was concentrated by precipitation by DEAE-cellulose chromatography. with (NH,)&lO,, dissolved in 1.0 ml of 50 mM-Tris.HCl (pH 7.5) and stored frozen at - 20°C. Wild-type (dam + ) and mutant (damh) forms of phage T2 DNA adenine methylase were purified by MS Joan Brooks and Dr Malthi Masurekar, respectively. (b) Isotopes and analytical
materials
&‘-adenosyl-L-[methyl-3H]methionine and S-adenosyl-L-[meth$-14C]methionine were obtained from Amersham/Searle. The 5’-deoxydinucleotides pApC, pApT, pApA and pApG were obtained from Collaborative Research, Inc., Waltham, Mass. 2’-Deoxyadenosine was purchased from Calbiochem. DEAE-cellulose paper (DE81) was purchased from Whatman, Inc. Autoradiographs were made on Kodak RR/R54 Royal X-O Mat or medical X-ray film; development was with Kodak D19. Acid fuchsin and methyl orange were purchased from Fisher Scientific; xylene cyan01 FF was from Eastman Organic Chemicals.
(c) Digestion. with pancreatic DNaee Non-glucosylated, non-methylated phage T2 gt- damh dam-l DNA (Brooks & Hattman, Radioactively labeled 1973) was used as the substrate for the dam + or damh methylase. DNA was prepared by in. vitro methylation using S-ndenosyl-L-[methyZ-3H]methionine or ,S-adenosyl-n-[~&hyZ-14C]mothionine as the methyl donor (see Table 1). The labeled extensively against water DNA was deproteinized by extraction w*itb pl~cnol, clialyzed
TZ DNA
METHYLASE
363
SPECIFICITY
and concentrated under a stream of air. The DNA was then hydrolyzed with (elect,rophoretically pure) pancreatic DNase I by one of two procedures. (1) The DNA solution was evaporated to dryness and then suspended in 50 ~1 of a 1.0 mg/ml solution of DNase I (in 10 mM-Tris.HCl, pH 7.6 plus 10 mM-Mgcl,). After incubation at 37’C for 18 h, an additional 25 ~1 of enzyme was added. Following 3 to 4 h further incubation, the hydrolyzate was either lyophilized or evaporated to dryness under a stream of air. DNA digested by this method was subjected to S-dimensional electrophoresis (Sanger & Brownlee, 1967). (2) The concentrated DNA was digested at 37°C for 18 h in a reaction mixture (5 ml) containing 20 mM-Tris *HCl (pH 7.6), 10 mM-MgCl, and 250 pg DNase I/ml; an additional 100 pg DNase was added/ml and incubation was continued for 3 to 4 h. The reaction mixture was either frozen at - 20°C or treated with E. toll: exonuclease I (see below-). (d) Exonuclease
I digestion
as described above (method 2), glycine buffer (pH 9.2) Following DNase I digestion was added to a final concentration of 0.1 M. Then 20 ~1 of E. coli exonuclease I was added and the mixture was incubated for 4 11 at 37°C. The digest was neutralized with HCI and then frozen at - 20°C until ready for use. (e) .B’ractionation
of oligonucleotides
accorchg
to chain
length
Frozen DNA digests were thawed and diluted lo-fold with distilled water. The sample was applied to a 1 cm x 11 cm column of DEAE-cellulose (in water) ; the column was washed with 3 bed vol. of 2.5 mM-sodium acetate (pH 4.8) in 7 M-urea; fractionation of oligonucleotides on the basis of chain length was achieved by elution with a 200-ml linear acetate (pH 4.8) and 7 M-urea (Tomlinson NaCl gradient (0.0 M to 0.20 M) in 2.5 mM-sodium $ Tener, 1963). Fractions (2 ml) were collected and portions were taken for radioactivity determination. The di-, tri- and tetranucleotide peak fractions were pooled and desalted (see below). Oligonucleotides of chain length 6 or longer were not eluted under these conditions. (f) Desalting of oligonucleotides Pooled oligonucleotide fractions of a given chain length were diluted 5 to lo-fold in distilled water. The sample was applied to a l-ml bed volume column of DEAE-cellulose (in water). Urea and salts were removed by washing the column with 20 ml water and then 20 ml of 0.02 M-triethylammonium bicarbonate (pH 8.5). The nucleotides were eluted with 2 ivf-triethylammonium bicarbonate (pH 8.5); the sample was concentrated under a stream of air, and then evaporated to dryness in vacua. The dried samples were twice resuspended in water and dried in vacua on Parafilm (American Can Co.) sheets. (g) Dephosphorylation
of oligonucleotides
with alkaline
phosphatase
Nuoleotides were eluted from DEAE-paper with 2 nr-triethylammonium bicarbonate (pH 9.5) and dried on Parafilm sheets in vacua; the samples were taken up in 10 ~1 of a 0.1 mg/ml solution of E. coli alkaline phosphatase (in 50 mM-TriseHCl, pH 8.9, plus 10 mM-Mgcl,) and incubated in drawn capillary pipets for 2 to 3 h at 37%. The reaction was terminated by spotting on DEAE-paper. (h) Digestion
with snake venom
phosphodiesterme
For complete digestion, dephosphorylated oligonucleotides were suspended in 10 ~1 of a 0.1 mg/ml solution of snake venom phosphodiesterase (in 50 m&r-TriseHCl, pH 8.9, plus 10 mM-MgCl,) ; incubation was carried out as described above (section (8)). Separate experiments showed that the enzyme preparations had no detectable 5’-nucleotidase activity. For partial digestion, the enzyme concentration was 25 pg/ml and the reactions were carried out for 0, 5, 10 and 30 rnirr at 22°C. (i) Digestion with spleen phosphodiesterase Just prior to use, spleen phosphodiesterase was diluted in, and briefly 0.05 M-ammonium succinate (pH 6.5), 1 mnr-Na,EDTA and 0.05yo (w/v)
dialyzed against,, Tween 80 (Fisher
364
8.
HATTMAN,
H.
VAN
ORMONDT
AND
A.
DE
WAARD
Scientific). For complete digestion, oligonucleotides were resuspended in 10 ~1 of spleen phosphodiesterasc (0.1 mg/ml), 2’-deoxyadenosine (1.0 mg/ml) and incubated for 4 h at 37°C. For partial digestions, the enzyme concentration was 10 &ml, and reactions were carried out as described in section (h), above. Since many commercial preparations of spleen phosphodiesterase are contaminated with an enzyme that deaminates deoxyadenosine, or demethylaminates P-mcthylet al., 1974a), carrier deoxyadenosine was added t,o protect deoxyadenosine (Dugaiczyk against loss of any labeled methylated nucleosides generated by phosphodiesterase cleavage. Our preparation of spleen enzyme, however, did not contain significant de-
aminating
activity.
3. Results (a) Fmctionation
of obigonucleotides
according to chain length
As a prelude to analyzing DNA oligonucleotides containing mA, it was desirable to fractionate enzymatic digests according to chain length (isostichs). One convenient method involves ion-exchange chromatography on DEAE-cellulose in the presence (Tomlinson & Tener, 1963). Figure 1 shows the chromatographic separaof 7 M-Urea tion of oligonucleotides produced following pancreatic DNase digestion of 32Plabeled (non-glucosylated) T2 gt- DNA. Pancreatic DNase produces relatively few mononucleotides (Sinsheimer, 1954); using a 0 M to 0.25 M-NaCl gradient, isostichs of up to chain length 7 could easily be discerned (Fig. l(a)). When the DNase digest
300 L
1
Co”+rol’
X
600
25
75
50
loo
25 Fraction
50
75 (b)
(0)
FIG. 1. Separation cellulose
digested cellulose
100
number
of 32P-labeled oligonucleotides of similar chain length (isostichs) by DEAE32P-labeled T2 gt- dam” darn-1 DNA was isolated and chromatography (in 7 M-urea). with pancreatic DNase (see Materials and Methods). The digest was subjected to DEAEchromatography (in 7 M-Urea) before and after further digestion with E. coli exouuclease 1
((a) and (b), respectively). The conditions and Methods section. The numbers indicate
for chromatography the chain
lengths
are described of the various
in the Materials
isostichs.
T2
DNA
METHYLASE
365
SPECIFICITY
was treated with E. co& exonuclease I prior to column chromatography, most of the 32P label was recovered in mononucleotides and dinucleotides (Fig. l(b)) ; some 32P label was still observed in the trinucleotide region indicating that a limit digest was not achieved. non-methylated) was used as the subT2 gt - darn” dam-l DNA (non-glucosylated, strate for in vitro methylation by the T2 dam+ and damh enzymes, respectively ; separate experiments verified that mA was the only labeled base produced. Figure 2 shows the chromatographic fractionation of [3H]oligonucleotides produced following successive treatment with pancreatic DNase and exonuclease I. The [3H]dinucleotide peak fractions were pooled, desalted and analyzed further (see below).
damh
dam+ I
I
2250
t = 2 2000 lo k za .z 1000 Lo Tl x. F t 750 8 a e ,I 500 -
3
3
I
250 L2 25
50
75
loo
25 Fraction
?( z 59
75
loo
number
(0)
(b)
FIG.
2. Separation of [3H]methyl-labeled isostichs by DEAE-cellulose chromatography. T2 gt- damh dam-l DNA was methylated in vitro by either (a) T2 dam+ or (b) T2 dam” methylase, digested successively with pancreatic DNase and E. co& exonuclease I, and subjected to DEAEcellulose chromatography (see Materials and Methods).
(b) Characterization
of N6-methyldeoxyaden~osine-containing
dinucleotides
The [3H]mA-containing dinucleotides were analyzed by high voltage electrophoresis on Whatman 3MM paper; four authentic deoxydinucleotide markers containing 5’-A were co-electrophoresed in the same track as the sample. At pH 1.9, three peaks of 3H radioactivity were observed (Fig. 3(a)). Two negatively charged [3H]dinucleotides co-electrophoresed with marker pApT and slightly ahead of marker pApG, respectively. The third [3H]dinucleotide migrated toward the cathode with
366
S. HATTMAN,
H.
VAN
ORMONDT
AND
il.
DE
WAARD
pApA and pApC (not resolved at pH i-9). However, at pH 3.5, all four marker dinucleotides were separated (Fig. 3(b)); it is clear that 3H label is in the pApC region and not in pApA. The 3H-labeled (MA*, G) and 3H-labeled (mA*, T) were not resolved under these conditions; the parentheses indicate that the sequence is not known. Similar results were obtained with the dinucleotides from both dam+ and dumb methylated DNA. The absence of 3H-labeled (mA*, A) is in excellent agreement with the observations of Vanyushin et al. (1971) who studied ma-containing purine tracts in T2 DNA; they found mA in the purine dinucleotide (mA, G), and not in (mA, A) nor (mA, mA). Complete digestion with spleen or snake venom phosphodiesterase showed that the sequences of the three dinucleotidesare G-mA*,mA *-hmC, and mA*-T (nucleotide sequences will be written from left to right to correspond to the 5’ to 3’ polarity). This suggests that the dam+ and damh methylases recognize sequences containing
(0)
’ pti 1.9
I
I
I
I
I
500 400
PAPG -
-
-
300
PAPT
-
200 100
-5
< 2 u ,: Ii x 2
b)
I
0
5
I
I
PAPC -
350
IO
I5
-20
I
I
I
PAPA -
-r
PAPG PAPT --
pH 3.5 300
I? 250 200 150 I00 50
5
IO 15 20 25 Distance from orgIn km1
I30
35
FIG. 3. Electrophoretic analysis of [3H]mcthyl-labeled dinucleotides from T2 gt- dam” dam-l dinucleotide fractions (see Fig. 2) DNA methyl&ted in vitro with T2 dam “. The [3H]methyl-labeled were pooled, desalted and subjected to paper electrophoresis (Whatman 3MM) at either (a) pH l-9 or (b) 3.6. Authentic deoxynucleotides of known sequence were included ES ultraviolet absorbs+nce markers. After the paper (2 cm x 67 cm) w&s dried, l-cm strips were cut and placed in scintillation vials; 0.5 ml water plus 6 ml scintillation fluid were added, and the 3H radioactivity W&B measured in a Packard scintillation counter.
T2
DNA
METHYLASE
367
SPECIFICITY
G-mA*-hmC and G-mA*-‘I’. We should point out that the ratio of 3H label in G-mA*/(mA*-T + mA*-hmC) was greater than unity; this is probably due to the fact that pancreatic DNase shows some cleavage specificity. It generally produces 5’-hmC rather than 3’-hmC-containing dinucleotides; i.e. there is a preponderance of 5’-hmC-N weraus 5’-N-hmC (van Ormondt & Hattman. 1976). This has also been observed with pancreatic DNase digests of C-containing DNA (Dugaiczyk et aZ., 19746; our unpublished observation). Thus, the recovery of mA*-hmC, but not hmC-mA*, indicates that the latter sequence is probably not present. (c) Two-dimensional
fingerprints
of [14C]methyl-labeled
oligonucleotides
In order to obtain more detailed information about the methylation sequence(s), it was necessary to examine larger ma-containing oligonucleotides. Previous studies from our laboratory established the two-dimensional fingerprint for hmC-containing, non-glucosylated T2 DNA (van Ormondt & Hattman, 1976). After in vitro methylation and pancreatic DNase digestion, the [14C]methyl-labeled oligonucleotides were ta,ken either directly for fingerprinting, or the di-, tri-, and tetranucleotides were first purified by DEAE-cellulose chromatography (using a 0 M to 0.20 M-NaCl gradient)
I
dam+
25
50
75
25
loo Fmction
(0 1
50
75
100
number (b)
FIG. 4. Separation of [14C]methyl-labeled isostichs by DEAE-cellulose chromatography. T2 gt- duw@ danz-1 bNA was methylated in vitro by either (a) T2 dam + or (b) T2 dam” methylsses, digested with pancreatic DNase, and subjected to DEAE-cellulose chromatography. The di-, tri-, and tetranucleotide peak fractions (indicated by braces in the Figure) were pooled, desalted and analyzed further (see text).
368
S. HATTMAN,
H.
VAN
ORMONDT
AND
A. DE
WAARD
a,nd then fingerprinted The combination of these two methods enabled us to assess more easily the nature of the various oligonucleotides. Figure 4 shows the chromatographic separation of [14C]methyl-labeled oligonucleotides produced by pancreatic DNase digestion of in vitro methylated T2 gt- damh dam-1 DN.4. The tri- and tetranucleotide regions were collected, desalted and subjected to two-dimensional electrophoresis. Fingerprints of the purified tri- and tetranucleotide fractions are shown in Figures 5 and 6, respectively. The nucleotides were eluted from the DEAE-paper, had their 5’-terminal phosphate groups removed by treatment with E. coli alkaline phosphatase, and were then fractionated further by ionophoresis on DEAE 81-paper at pH 3.5 ; in most instances, another ionophoretic fractionation was carried out at pH 3-5 and/or pH l-9. This procedure allowed us to sepa.rate a variety of sequence isomers and compositionally related oligonucleotides that are barely resolved in the two-dimensional fingerprint (Sanger & Brownlee. 1967). The sequence of each nucleotide (mixture) was then determined by partial hydrolysis with spleen and snake venom phosphodiesterases (see below).
(d) Sequence asalysis
of labeled N6-methyldeoxyadenosine-containing
tGnuc1eotide.s
Table 1 contains a summary of the sequences deduced for the mA*-containing trinucleotides (Fig. 5). The base composition of each nucleotide could be inferred, in part, from its relative position in the two-dimensional fingerprint, and the R, values? (following dephosphorylation) on DEAE-paper at pH 3.5 and 1.9 (knowledge of the mA*-dinucleotides was also a major aid in this analysis). The sequence of nucleotide 1 must be mA*-hmC-hmC because, in addition to bhe above criteria, we know that mA*-hmC and not hmC-mA* is observed among the mA* dinucleotides (see section (b), above). It is important to note here that, under the conditions of venom exonuclease digestion, no partial degradation products were seen. It was subsequently observed that all oligonucleotides containing a 3’-terminal hmC residue were relatively resistant to venom exonuclease digestion. This property proved to be a useful diagnostic tool, as will be discussed below (for nucleotides 3 and 4). Nucleotide 2 is a minor species that is barely visible in Figure 5. Based on the criteria outlined above. we believe the only sequence possible for it is mA*-hmC-A. Nucleotide 3 appears to be a mixture of mA*-T-hmC and mA*-hmC-T; the absence of mA*-T as a partial digestion product is attributed to the presence of the 3’-terminal hmC residue. Nucleotide 4 appears to be primarily hmC-G-mA* ; however, since venom exonuclease digestion never completely degraded the starting material, we believe that the G-mA*-hmC sequence isomer is present. This would be consistent with the observed dinucleotides, G-mA* and mA*-hmC (and with the observed tetranucleotides, G-mA*-hmC-hmC and G-mA*-hmC-T (see section (e), below). Included in nucleotide 4 was another minor species which was not analyzed: it is likely that this is the mA*-hmC-G isomer. Trinucleotides 6 through 8 could be established with little ambiguity. Nucleotide 9 is present as a minor component (note: appears to be mainly T-G-mA * ; G-mA*-T G-mA*-T was expected to be a major species, since G-mA* and mA*-T are major dinucleotides). mA*-T-G is also a minor component of spot 9. We concluded that t IZB value is the distance
migrated
by oligonuclcutide/distancc
migrated
by blue dye marker.
60
70
(b)
03
04
<:35
f
8.D.
-+E-
FIG. 5. (a) Radioautograph of a 2-dimensional ionophoretic separation of trinucleotides purified from a pancreatic DPl‘ase digest of phage Td gt- dum” and [14C]mothyi-labeled S-adenosyl-I,-mcthionine. dam-1 (non-glucosylated, non-methyleted) DNA that had been methyl&& with the T2 dam + methyl&se In the first dimension, ionophoresis was on cellulose acetate at pH 3.5 (pyridine/acetato buffer); aftor transfer to DEAE 81.paper, ionophoresis in the second dimension was at pH 1.9. The hatched zone indicates the position of the blue dye, xylene cyan01 FF (spot,ted on both sides of the origin on cellulose acetate). Film exposure was for 9 days. (b) Diagram of the radioautograph. The numbered spots correspond to the oligonucleotide numbers given in Table 1. Broken lines indicate minor spots; thin lines indicate intermediat.e spots and thick lines indicate major spots. P.1). and B.D. refer to the pink dye (acid fuchsin) and blue dye, respectively.
IO 98
(0)
(b)
FIG. 6. (a) Radioautograph of a 2-dimensional ionophoretic separation of totranucleotides purified from a pancreatic DNase digest of phage T2 gt- damh dam-l DNA that had been methylated with the T2 darn” enzyme (see the legend to Fig. 5). Film exposure was for 7 days. (b) Diagram of the radioautograph.
1.9
1.31 1.35 2.45 2.41 2.00 N.D. N.D. 2.35 1.96 1.52 1.65 1.76
RB values$ 3.5 pH
1.40 1.35 1.45 1.39 1.20 1.10 0.47 1.15 1.00 0.90 1.10 1.20
pH
trilsucleotides
gt-
mA*
dig&ion products Spleen
phage T2
N.D. N.D. mA*p mA*p mA* N.D. N.D. mA*p Gm-A*; mA* mA*; G-mA* mA*p; G-mA*; mA*p
Partial
from with
damh
DNA
None N.D. None mA*-hmC pmA* N.D. N.D. mA*-T; mA* pmA* pmA* pmA*; G-mA*; mA*-T; mA*
phosphodiesterase Venom
dam-l
1
mA*-T
from:
§ trinucieot.ide
sequence?
methylase
mA*-hmC-hmC mA*-hmC-A mA*-T-hmC mA*-hmC-T hmC-G-mA*; (G-mA*-hmC) (mA*-hmC-G) (?) Tetranucleotide mA*-T-A A-G-mA* G-G-mA* T-G-mA*; (G.mA*-T; mA*-T-G) mA*-T-T
Deduced
modi$ed with T2 DNA
Non-glucosylated, non-methylated T2 DNA (from T2 gt- dam” dam-l) was methylated in vitro with purified preparations of DNA adenine methylase induced by wild-type T2 phage (dam +) or mutant (damh) using [14C]methyl-labeled S-adenosyl-n-methionine as the methyl donor. 3000 units of T2 dam+ enzyme were added to a standard reaction mixture (Brooks et al., manuscript in preparation) containing 250 pg of substrate DNA. After 60 min of incubation at 37”C, an additional 1500 units of enzyme were added; after a further 60.min incubation period, the DNA was extracted and dialyzed against water. A similar reaction was carried out in parallel, with 1500 units of T2 dam” methylase and 200 pg of DNA (300 units of dam” enzyme was added after the first 60-min incubation period). The stoichiomet)ry of methylation at saturation was calculated to be 3000 and 1500 methyl groups added per phage DNA by urn” and dam + , respectively. t The nucleotidc numbers are t.hose shown in Fig. 5(b). The 2.dimensional trinucleotide fingerprints for damh and dctm + were identical. 1 Following dephosphorylation with alkaline phosphatase, relative ionophoretic mobilities (RB) were measured with respect to the blue dye, xylene cyan01 FF, by eleotrophoresis on DEAE 81.paper at pH 1.9 and pH 3.5; thedye was allowed to migrate at least 12 cm from the origin. The figures listed are average values; generally, the variation was -C 10% of the mean value and, in a given experiment, t,he 11, values tended to be uniformly higher or lower than the mean value. N.D., not done. 5 The partial digestion products were identified from their ionophoretic mobilities on DEAE 81.paper at pH 3.5 and pH 1.9. These were also compared of 0.4 to 0.5). Since the dinuoleoside monoto values for a2P-labeled oligonucleotides which lacked mA (at pH 3.5, the deoxynucleoside, mA *, has a mobility phosphate, G-mA* , and the mononucleotide, pmA* or mA*p, have similar mobilities at pH 3.5, it was necessary to analyze these further. (The 3’, 5’ phosphodiester bonds will generally be represented by dashes ; terminal 3’ or 5’-phosphates will be indicated in the appropriat,e positions.) This was carried out to flatby one of two methods. (1) Aftes autoradiographic analysis and location on DEAE-paper, the spots (Es = 1.8 to 1.9) were eluted and subjected plate electrophoresis on (2.5 cm x 57 cm) Whatman 3MM paper (0.1 M Tris, pH 8.0) to separate the mononucleotide and dinucleoside monophosphate. Authentic markers were co-electrophoresed and located by their ultraviolet light absorbance; l-cm width strips were cut and the r*C radioactivity counted. (2) DEAE-paper electrophoresis at pH 1.9 also proved to be a convenient method, since, at this pH, G-mA* migrates much slower than pmA* or mA*p. N.D., not done. 11 The minor species observed are enclosed in parentheses. See text for discussion.
1 2 3a 3b 4a 4b 5 6 7 8 9 10
Sucleotidet number
Methylated
TABLE
mA*.‘F.‘r.
mA*p;
G-G.mA*;
0.68
0.7’1
0.35
10b
1oc
118
G-m&4*;
mA*-T-hmC mA*
analyzed
pmA* mA*-(T, hmC) pmA*; G-mA*; hmC-G-mA* mA*-T; pmA*;
Not
A-G-mA*; G-mA*; mA* mA*p mA*; mA*p; m.4*-(T, hmC) (T-G-mA*/G-m.4*-T) mA*p; hmC-G-mA*/G-mA*-hmC
hmC)
0.43 047 0.59
mA*-(T,
mA*p
modijied
pmA*
mA*-hmC-A
mA*.‘p
mA*-T-hmC
G-mA*-hmC
mA*-hmC-A mA*-T; mA*-(T,hmC) mA*-hmC or mA*-hmC-hmC t unidentified products mA*-T-hmC G-mA*-hmC
NOW
NOM!
8 9 10a
mA*p;
;
DNA
phosphodiest,erase from?: Venom
mA*p mA* -hmC-hmC
with
o.ti4 0.76 0.72 0.73
produck
5a 5b s I
digestion Spleen
damh dam-l
8.D. N.D. N.D. m.4*p
Partial
from phage T2 gt-
0.93 0.86 0.63 0.85
R, valuet at pH 3.5
tetranucleotides
1 2 3 4
Nuoleotide numberf
Methylated
TABLE 2
tetranucleotido
methylasei sequence
3
A-G-G-m-4*
mA*-T-hmC-A G-mA*-hmC-hmC (G-mA*-hmC-A/A-G-mA*-hmC) (?) mA*-hmC-A-G + dephosphorylated G-mA*-T-hmC A-A-G-mA* mA*-(T, hmC)-G or mA*-(T, hmC)-;l-P hmC-G-mA*-T; hmC-T-G-mA*; G-mA*-hmC-T mA*-T-hmC-(T or G) T-G-mA*-hmC/T-hmC-G-mA* G-mA*-T-hmC; mA*-1’.T-hmC
mA*-hmC-hmC-hmC mA*-hmC-A-hmC m-4*-hmC-A-A mA*-(T, hmC)-hmC’ mA*-hmC-T
Deduced
with T2 DNA
-
mA*-T-A; 4.G-m4* ‘r-G-mA*/G-mA*-T; mA*p; mA* T-G-mA*/G-mA*-T; A-G-mA*; G-mA* ; mA*-T; mA*p; mA* G-G-mA*; T-G-mA* G-G-mA*; mA*-T G-mA*-T ; mA*p mA*-T-T; G-mA* mA*-T-T; G-mA* T-G-mA*; mA*-T mA*; G-G-mA* mA*p mA*-T; mA*-T-T G-mA*; mA*-T-A; ; mA*
T-G-mA* ; pmA*
mA*-T-T; mA*-T; T-G-mA*/G-mA*-T
G-mA*-T; G-G-mA*
pmA* G-G-mA*; pmA* mA*-T; G-mA*
mA*-T-T; A-G-mA*
mA*
T-G-mA*/G-mA*-T/mA*-T-G G-mA*; pmA*; mA*-T
mA*-T-T-G; G-mA*-T-T
mA*-T-T-G; G-G-mA*-T; T-T-G-mA*;
mA*-T-A-G mA*-T-G-A
A-G-mA*-T; mA*-T-T-A’ T-A-G-mA*’ G-G-G-mA* G-G-mA*-T; mA*-T-G-(G
T-G-mA*-T
G-mA*-T-T T-G-mA*-T T-G-G-mA*
; G-T-G-mA* T-G-G-mA* or T); G-mA*-T-T
mA*-T-G-A ; G-mA*-T-A
T-A-G-mA*; A-T-G-mA*
t See the legend to Table 1. Some of the tetranucleot,ides were not detected in the fingerprint (Fig, 6); however, they were obtained from an independent preparation and are included in the Table. 1 The nucleotide numbers are those shown in Fig. 6(b). The 2.dimensional tetranucleotide fingerprints for dam” and dam+ were identical. product, from 5 The base composition was inferred from the position of the oligonucleotide in the fingerprint, from the R, value of the dephosphorylated the products of (or resistance to) partial exonuclease digestion, and from the observed mA*-containing trinucleotides (Table 1). This strategy, however, did not always lead us to an unambiguous sequence assignment. In cases where we could not distinguish between sequence isomers, we have either enclosed the relevant bases in parentheses or written both sequences, connected by a slash (c.g. nucleotide lob could contain T-G-m-4*-hmC and/or T-hmC-G-mA*). N.D.. not done.
0.48 ; 0.53
13b
0.46
0.33 0.40
12a 12b
0.43;
0.49
llc
13a
0.4”
llb
374
S. HATTMAN,
H.
VAN
ORMONDT
AND
A. DE
WAARI)
nucleotide 10 is mA*-T-T, and not mA*-T-G, due to its position in the fingerprint relative to G-G-mA* and T-G-mA*, and to its higher R, value (at pH 3.5) than T-G-mA*; for example, since sequence isomers with a 5)-A residue tend to have a somewhat lower R, value than their counterparts containing a 3’-A residue (Sanger & Brownlee, 1967 ; our unpublished observations), if spot 10 were mA*-T-G, it, would not have had a higher R, value than T-G-mA*. Finally, the observa,tion that G-mA*-T-T is among the mA*-tetranucleotides (see section (e). below) is consistent with the occurrence of mA*-T-T. Based on the observed dinucleotides, mA*-hmC, mA*-T, and G-mA*, we had anticipated that G-mA*-T and G-mA*-hmC would be among the major mA*trinucleotides. As shown in Table 1, these sequences represented only minor species ; mA* was observed mainly at the 5’ or 3’ position of the trinucleotides characterized. It is possible that the N6-methyl group of mA* exerts some influence on the specificity of pancreatic DNase cleavage. From the results summarized in Table 1, it appears that a variety of methylated trinucleotide sequences are produced by both dam+ and da,mh; namely X-G-mA*, G-mA*-Py, and mA*-Py-N. The only species for which there is some uncertainty is mA*-hmC-G. Nevertheless, it appears that the recognition sequence for both enzymes is simply N-G-A-Py-N: however, we do not believe that all these sites can be methylated with equal ability by dam+ and damh (see Discussion). (e) Xequence analysis
of tetranucleotides
tetranucleotides obtained Figure 6 shows a fingerprint of purified [‘“Cl methylated after pancreatic DNase digestion and DEAE-cellulose chromatography of T2 gtdamh dam-l DNA methylated by the T2 damh enzyme (see Materials and Methods). Table 2 contains a summary of the sequence analysis of the various nucleotides. As with the trinucleotides, we observed no reproducible, significant differences in the DNA ; fingerprints of tetranucleotides from T2 damh veraus T2 dam+ methylated furthermore, due to the larger number of sequence isomers in each spot, compared to trinucleotides, it was even more difficult to detect any quantitative differences among tetranucleotides. Based on the general methylation sequence, 5’. . . N-G-mA*-PyN.. .3’, derived from the trinucleotide analysis, there are 64 possible tetranucleotide sequences. We observed approximately 213 of t,hese combinations, which are likely to correspond to the most frequently occurring methylated tetranucleotide sequences in T2 DNA. The observation of tetranucleotides containing G-mA*-hmC and GmA*-T (nucleotides 5b, lOa, b, c and lib, c) confirmed these trinucleotide assignments.
4. Discussion Although the cleavage site for many site-specific endonucleases has been established, only a relatively small number of DNA methylation sequences have been elucidated. In practice, the determination of methylation sites is a more tedious procedure. This generally starts with the in vitro methylation of a substrate DNA with (partially) using either [14C]- or [3H]methyl-labeled Spurified DNA methylase activity, adenosyl-L-methionine as the methyl donor. The DNA is then enzymatically degraded and the resulting oligonucleotides are fractionated and sequenced. The methylation sequence is deduced from the overlapping sequences determined for the various site is a unique sequence of radioactive oligonucleotides ; when the methylation
T2
DNA
METHYLASE
375
SPECIFICITY
four to six residues, the analysis is simplified by the presence of only a small number of labeled nucleotides. Using this general approach we have investigated the site(s) methylated by the dam + and da+ forms of the T2 DNA adenine methylase. The damh enzyme methylates two- to threefold more sites than the dam+ enzyme; e.g. approximately 3000 and 1000 mA residues per mature phage T2 gt - DNA are produced by damh and darn, + , respectively (Hattman, 1970). (We estimated that the dam+ methylation sequence should be about four to five (specific) nucleotide residues long.) Thus, we hoped that our analysis would reveal mA-containing oligonucleotides unique for damh methylation. Contrary to this expectation, we observed the same methylated di-, tri- and The methylated sequence tetranucleotides for the dam + and damh methylases. consistent with these oligonucleotides is 5’. . . N-G-mA*-Py-N . .3’. Further support for this comes from the level of in vitro methylation attained; e.g. 3000 mA residues per T2 DNA (see Table 1) corresponds to about 300;;, of the calculated number of G-A-Py sequences (based on random distribution of bases in T2 DNA). Recent experiments have shown that it is possible Do reach higher methylation levels and approach the theoretical maximum (unpublished data). The results presented extend those reported for T2 dam+ methylation of micrococcal DNA, where the only sequence methylated was G-mA*-T-C (van Ormondt et al., 1975). Tt is not clear why we observe a lower methylation specificity with T2 gl- DNA; it is not due to the presence of hmC in T2 phage DNA, since we also find G-mA*-C in h DNA methylated by T2 dam+ (unpublished observations). Tt should also be noted that the occurrence of certain methylated tetranucleotides (e.g. G-mA*-hmC-hmC, G-mA*-T-T and G-mA*-T-A) indicates that S-fold rotational symmetry is not required for sequence recognition. This has been observed with the et al., 19740) M.EcoB modification methylase (van Ormondt et al., 1973; Dugaiczyk and the M.EcoPlf modification methylase (S. Hattman. J. Brooks & M. Masurekar: manuscript in preparation). Our analysis failed to reveal any qualitative difference(s) in methylation specificity between T2 dam+ and T2 damh; furthermore, we did not observe any significant. reproducible quantitative differences in the occurrence of mA*-oligonucleotides (as judged by visual inspection of autoradiograms). However, a failure to detect any qua’ntit’ative difference might have been the result of selecbive degradation during pancreatic DNase digestion. This is a plausible explanation for the low recovery of mA*-hmC, G-mA*-hmC and G-mA*-T; we have also observed this with methylated C-containing oligonucleotides (unpublished data). Therefore, it is difficult to assess to what extent pancreat’ic DNase cleavage specificity might have been a factor in our failure to detect qualitative or quantitative differences between T2 dam+ veTsus T2 dam,h methylation. Although both methylases produced G-mA*-Py, it is possible that t,he two enzymes do not methylate all sites with equal ability (further studies are in progress). In this sense, dam+ might have a low affinity for certain of the G--A-Py sequences; the mutation to damh might result in an altered enzyme which can methylate the “low affinity” sites more readily. A change in nucleotide sequence recognition has been observed under special conditions in other systems. It is known that ccrt’ain restriction enzymes do not cleave all sites at the same rate (Thomas $ t ~l~Ec01’1 is t,he modification methylase used to identify restriction and modification 1973).
specified by phage Pl (the enzymes is that suggested
three-letter by Smith
designation & Nathans,
876
S. HATTMAS,
H.
VAN
ORMONDT
AiWI
A. DE
WAARD
Davis, 1975) and that in vitro substrate specificity can be lowered by varying the pH and ionic conditions (Polisky et al., 1975). In the case of the T2 methylase forms, the change in sequence specificity is the result of a mutational alteration; it remains to be seen whether it will be possible to obtain similar mutants for other DNA methylases. Finally, the damh mutant was originally isolated from T2 gt - dam + as being resistant to restriction by phage Pl, The Pl-modification enzyme has been reported to produce the sequence 5’. . A-G-mA*-T-C-T.. .3’ (Brockes et al., 1974) : this sequence should be recognized by both dam + and dam”. Recent studies, however, indicate that the MS EcoPl sequence is 5’. . . A-G-mA*-C-Py . .3’ (S. Hattman, J. Brooks & M. Masurekar, manuscript in preparation) and that this site may be one that is not readily methylated by dam+ (J. Brooks, S. Hattman & M. Masurekar, manuscript in preparation). We grat,efully acknowledge the assistance of ,I. Brooks and M. Masurekar for their role in purifying the T2 methylases, the production of in vitro mrthylated DNA, and for mana critical discussions. We also acknowledge the participation of J. Garter in the early phases of the sequence analysis. This work was supported by a Public Health Service grant (no. AI-10864) and a Research Career Development award (no. AI-28022) to one of us (S. H.). REFERENCES Brockes, J. P., Brown, P. R. & Murray, K. (1974). J. Mol. Biol. 88, 437-443. Brooks, J. & Hattman, S. (1973). l’irology, 55, 285.-288. Dugaiczyk, A., Boyer, H. W. & Goodman, H. M. (1974a). Biochem. Biophys. Res. Commun. 56, 641-646. Dugaiczyk, A., Kimball, M., Linn, 8. & Goodman, H. M. (19746). Biochem. Biophys. Res. Commun. 61, 1133-1140. Dunn, D. B. & Smith, J. D. (1958). Biochem. J. 68, 625-636. Fujimoto, D., Srinivasan, P. R. & Borek, E. (1965). Biochemistry, 4, 2849--28X. Gefter, M., Hausmann, R., Gold, M. & Hurwitz, ,J. (1966). J. Biol. Chem. 241, 1995-2006. Gold, M., Hausmann, R., Maitra, U. & Hurwitz, ,I. ( 1964). I’roc. Nat. Acad. Sci., U.S.A. 52, 292-297. Hattman, S. (1970). virology, 42, 359-367. Hausmann, R. & Gold, M. (1966). J. Biol. Chem. 241, 1985-1994. Hehlmann, R. & Hattman, S. (1972). J. Mol. BioZ. 67, 351-360. Lehman, I. R. & Nussbaum, A. L. (1964). J. Biol. Chem. 239, 2628-2636. Polisky, B., Greene, P., Garfin, D. E., McCarthy, B. J., Goodman, H. M. & Boyer, H. W. (1975). Proc. Nut. Acad. Sci., U.S.A. 72, 3310.-3314. Revel, H. R. & Hattman, S. M. (1971). l’irology, 45, 484-495. Revel, H. R. & Luria, S. E. (1970). Annu. Rev. Genet. 4, 117-192. Revel, H. R., Hattman, S. & Luria, S. E. (1965). Riochem. Biophys. Res. Commun. 18, 545-550. Sanger, F. & Brownlee, G. G. (1967). In Methods in Enzymology (Grossman, L. & Moldave, K., eds), vol. 10, pp. 361-381, Academic Press, New York. Sinsheimer, R. L. (1954). J. BioZ. Chem. 208, 445-459. Smith, H. 0. & Nathans, D. (1973). J. Mol. BioZ. 81, 419-423. Sulkowski, E. & Laskowski, M. Sr. (1971). Biochim. Biophys. Acta, 240, 442-447. Thomas, M. & Davis, R. W. (1975). J. Mol. Biol. 91, 315-328. Tomlinson, R. V. & Tener, G. M. (1963). Biochemistry, 2, 697-702. van Ormondt, H. & Hattman, S. (1976). Anal. Biochem. 74, 207-213. van Ormondt, H., Lautenberger, J. A., Linn, S. & de Waard. A. (1973). FEBS Letters, 33, 177-180. van Ormondt, H., Gorter, J., Havelaar, K. J. & de Waard, A. (1975). Nucl. Acids Res. 2, 1391-1400. Vanyushin, B. F., Buryanov, Ya, I. & Beloeersky, A. N. (1971). Nature New BioZ. 230, 25-27.