In vitro methylation of bacteriophage λ DNA by wild type (dam+) and mutant (damh) forms of the phage T2 DNA adenine methylase

.I. NOT. Biol. (1978) 126, 381-394

In vitro Methylation of Bacteriophage I DNA by Wild type (dam +) and Mutant (damh) Forms of the Phage T2 DNA Adenine Methylase JOAS EBROOKS

AND STANLEY HATTMAN

The University of Rochester Depurtment of Biology Rochester, N.Y. 14627, U.X.A. (Received 13 December 1977, and in revised form

X Augwt

197X)

The wild-t,ype (dam+) and mutant (rZamh) forms of t’he bacteriophage ‘I’d DNA adeninc methylase have been partially purified; these enzymes methylate the sequencr, 5’ . . . G-A-Py . . . 3’ (Hattman et al., 197%). However, ain vitro lnet,hylation studies using phage X DNA revealed the following: (1) T2 dam + and rEamh enzymes differ in t’heir abilit’y to methylat,r h DNA; under identical reaction caondit.ions the T2 damh enzyme metjhylated X DNA to a higher level than did the

tlum + enzyme. However, the respective methyl&ion &es are equally distributed on t,hr I and T strands. (2) Methylation with T2 dam”, bnt not T2 dam+ protectc,d h against, Pl restrict’ion. This was demonstrated by t,ransfect’ion of Escherichin coli, (Pl) sphcroplasts a,nd by cleavage with R’ EcoPl. (3) T2 dum+ and rlnrrr” were similarly capable of met,hylating G-A-T-C sequences on /\ DNA%; ‘.g. h~lanl~ DNA (contains no Ne-mcthyladonine) methylatcd wit,h either enzyme was madr txistjant to clea,vaga by R. DpnII. In contrast, only the 7’2 damh modified DNA was resistant to further met,hylation by M*EcuPl (which methylates t,he sequence S’... A-G-A-C-Py . . . 3’; Hatt)man et al., 1978b). (4) X. rlum, DNA was partially metjhylatetl t,o t,he same level with T2 dam+ or ‘I’2 dnmh; the two enzymes proclucrd tiiffcrcnt patterns of G--4-C versw G-A-T methylation. We propose that, t,11c ‘1’2 da?,l+ cnzymp methylat,es G-A-C srqwnCes less &iciently than t,htb T2 rl~rr,~ mcthylase; this property does not cnt.irely wrcount. for the large tlifferenw in methyl&on levels produced by the two enzymes.

1. Introduction ‘I’2 ba&eriophage (as well as T4 and T6) differs from all other organisms by hydroxymethylcyt~osine in place of cytosine in their DNA (Wyatt & Cohen, ‘l’hc hmCyt7 residues are normally glucosylated by a phage-induced glucosyl fcrase enzyme (Lehman & Pratt, 1960). The glucose prot,ects t’he phage DNA

having 1952). kansagainst

i Abbreviations usrd: hmCyt, 5.hydroxymethylcytosiine; Me, adenine; MeAdr, N6-methyladoIline: L3H].4doMet or [14C]AdoMet, S-adenosyl-L-[meth~Z-~H or “C]methionino; J’MSF, phenvlTES, N-Tris (hydroxymethyl) methyl-2.aminoethane sulfonic a&l: methyl snlfonylfluoride; PIPES, piperazine-N-N’-bis (2.ethane sulfonic acid); M.EcoPl/R .EcoPl, modification methylase/ restriction endonuclease activities specified by phage Pl (the designations are those suggestrd by Smith & Nathans, 1973); h.(Pl), Pl-modified bacteriophage h. Since all X DNA preparations used in these studies were growrr in E. coli K strains, we have omitted designation of the K-specific modification: i.e. X. K is denoted simply as h, and A. K( PI ) as X.(Pl). 381 0022 -2836/78/%503X1- 14 $02.00/O

i(i) 1978 Academic

Press Inc. (London)

Lttl.

382

J. E. BROOKS

AND

8. HATTMAN

a variety of nucleases, including the Pl restriction endonuclease (Hattman, 1964; Revel, 1967; Revel & Luria, 1970). Phage mutants thah are non-glucosylated (gt) (Revel et al., 1965) are unable to grow on Pl lysogens of Escherichia cc& (Georgopoulos, 1969; Klein, 1965). However, T2 gt phage can mutate in a single step to a form which is less sensitive to Pl restriction. These mutants were originally designated uP1 (unrestricted by Pl) (Molholt, 1967; Revel & Georgopoulos, 1969). It was suggested that the uP1 mutation occurred in the structural gene for the DNA adenine mcthylasc of the phage (Hattman, 1970; Revel & Hatt’man, 1971; Hehlmann & Hat)tman, 1972). Concomitant with conferring Pl resistance, the rnut,ation increases the level of phage DNA methylat,ion two- t,o t’hreefold above t*he wild-t,ype level (Hattman, 1970). Therefore, when t,he gene was named dam (for DNA adenine methylaee), t’he mutanb was denoted by t)he superscript, “h” for hypermethylat,ion abilit’y (Brooks & Hattman, 1973). The dam+ and damh enzymes were compared with respect bo their abilities to methylate various DNA substrates (Revel & Hattman, 1971; Hehlmann & Hatt’man, 1972). The T2 damh enzyme introduced at least two t,o three times as many methyl groups on hmCyt-containing DNA as did t’he wild-type enzyme. (This was independent’ of whether the hmCyt was glucosylated or not.) However, in cytosine-containing DNAs (calf thymus or micrococcal), no differences in methylation levels were observed. These results cannot be att,ributed to the presence of hmCyt in T2 gt DNA because in vitro methylation of cytosine-containing T2 DNA was indistinguishable from that of hmCyt-containing T2 gt DNA (Hattman, 1972). Thus, it was postulated t,hat T-even phage DNA contains a large number of damh-specific sites that’ are missing (or present at reduced levels) on het,erologous DNAs (Hehlmann & Hattman, 1972). To test this hypothesis, it was of interest t’o extend this investigat)ion to another cytosine-containing DNA. Phage X DNA was chosen for t,his study because, like non-glucosylated T2 phage, it is subject to Pl rest,riction (Lederberg, 1957: Arber & Dussoix, 1962) and, hence, should contain damh-specific sites. This paper will describe the meOhylation of h DNA by the T2 dam+ and damh enzymes, and the relat,ionship between these methylases and the PI restriction-modification syst,em.

2. Materials and Methods (a) Bacterial Escherichia

coli

and phage strains

strains

1100 and llOO(Pl), and bact,eriophages T2 gt dam+ and T2 previously (Brooks & Hattman, 1973). E. coli strain K704 the dam, mutat,ion was was obtained from B. de Groot; E. coli strain GM48 containing obtained from M. Marinus; phago A~1857 was obtained from B. Dottin.

gt damh have been described

(b) Media and chemicals 1970). DNA-agarose was prepared L broth has previously been described (Hattman, by the method of Schaller et al. (1972). Calf thymus DNA and agarose were purchased from Sigma. Carboxymethyl Bio-gel was purchased from Bio-Rad. [14C]AdoMetj ( >40 mCi/mmol), [3H]AdoMet (5 to 15 Ci/mmol), [‘W]AdoMet (0.5 mCi/mmol) (the spec. act. is sufficiently low so that DNA preparations methylated with this methyl donor contained very little radioactivity; consequently, we utilized this in place of unlabeled AdoMet and it is indicated as unlabeled in t,he text) and the t’issue solubilizer (NCS) were obtained from Amersham/Searle; [2-3H]adenine (15 to 30 Ci/mmol) and [32P]orthophosphoric acid (carrier-free) were from New England Nuclear Corp. DpnI and II restrict,ion enzymes were a gift from S. Lacks.

T2 dam

METHYLATION (c) DNA

OF X I)NA

Xi3

preparutionn

E’. c*oZi st,rains 1100, llOO(P1) and X’. c&i dam, lysogenic for Xc1857 were grown in 1 1 01 I, 1)roth at 32°C to a titer of 4 x lOa cells/ml (determined microscopically). The cells w~r(~ barvested (10,OOOg for 10 min at 0%) and resuspended in 20 ml of L broth. To induce th
methylase

reactk

The T2 tlaw + or dawh m&hylase reaction mixt*ure (0.125 ml) contained: 0.05 M-I’ris.HC’l (l)H X.0), 0.008 M-EDTA, 0.15 ~Nacl, 22.8 pM-[14C]AdoMt’t or 13H]AdoMet,, 100 pg calt t hymns or h DNA/ml, and varying amounts of enzyme. Any changes in concentration of wact;mt,s arc noted in the text,. Aftjar incubation at 3O”C, portions from bhr reaction trlist,uros were precipitated in 5($/Atrichloroacctic acid and kept’ for 30 min at the ice I)ath i rrnpcratllre. The precipitate was collected by filtrat’ion onto Whatman GFjA glass filtvl, tlisks and xvashed with 12 ml of 1:; tzichloroacet,ic acid. Thr disks ww dried and their rntlioact,ivity mcasarcd in a Packard liquid scint,illation counter. One unitj of DNA twthylasc is defined as that, amount, of enzyme which, under the st,andard assay conditions. t ransf(>rx on,> pmol of methyl groups from AdoMot to calf thymus DNA in oncl Inin. (e) i’urijcntion

of the T2 DNA

methylases

E. coli K704 crlls were grown in L broth at 37°C to mid-log phastl (6 ,Y 10R cells ml, (k~tcrmined microscopically) and then infected wit,h a multiplicity of 5 phage (7’2 gt tlrcm, + or T3 gt tlnmh) cell. At 18 min post-infection, t.he ~11s were cooled I)y the addition of rrushed ice a,nd t,hcn harvested by centrifugation (10,OOOg for 10 min); tho pellet ~-as procedures in the purification were condurt,wI frozen and st,owd at -. 20°C. All suhsqucnt, at> 4’C. DNA-free crutl~ extracts were prepared by t,he procedurr of Reubw & Goftcr (1974): 10 g of thawfd cells were resuspended in 20 ml of bufftl in buffiir A containing 0.5 M-NaCl. Fractions (2.5 ml) were collected a,ntl assayc~l for nwthylnsc actirit,y; the act’ive fractions were pooled and dialywd overnight, against 2 1 0.02 nr-sodium phoaphatc (pH 7.0), 0.005 M-EDTA, 0.005 &I-2-mercaptoet.hano]. lO”,, glycerol (buffer B). The dialysat,e was applied to a c~arboxymethyl Bio-gel column (I.1 (‘rn ‘(5 cm) previously equilihratctl wit,h buffer B. After washing with 50 ml of Ollff~Lr B, a 100.ml linear gradient of NaCl (0 RI to 1.0 ill) in ln&‘er B was applied to th,. cwlutnn and 1.5-ml fractions collected. Methylasc activity c,lllted brtjween (+()fj :m(l 0.13 >I-NaCl. The active fractions were pooled, tlialyzwl against 1 1 0.02 iM-Tri$. H(‘l (pH K.O), 0.005 M-EDT& 0.005 M-2-mercaptoet,htmo], loo, glycerol, and stowd at 4 (‘. ‘lk~ dam + and rln,jlh methyluse activities were purified approximately SO-fold by this prowdllrr. They exhibited identical chromatographic beha\.ior. Under the DNA methylasc, assa,v conditions, both enzyme preparations were frw: of tlctectabltb nuclcaw activity, its moasrnwl by acid-solubiliza,tion of ?!?-labeled X DNA and by sedirncntat ion in ncwtral

384

J. E. BROOKS

AND

S. HATTMAN

sucrose gradients. Less than 1 “/b of the methylase activity present can be at,tributed to the host E. coli methylase; methylase purified from uninfected 1~11s showed tlistin&ly different, chromatographic properties when compared to t,he phage enzymes. Protein concont,rat’ion was determined by t,he method of Lowry et al. (1951). (f)

Transfection

vj’ E. coli spheru~lasts

hy in vitro

methylaterl

,I DNA

15 pg of h DNA were methyla,ted for 2 h in a O.25-ml rract,ion mixture containing 150 units of dam+ or danah methylase (2000 units/mg for each enzyme) and [l%]AdoMet as the methyl donor, An equal vol. of water was added and the mixture was heated t,o 65°C for 5 min (to disaggregate h cohesive ends). The samples were layered onto ll-ml linear sucrose gradients (5qi, to 20th) in 0.02 M-Tris.HCl (pH 7.5), 0.02 M-Na,Cl, 0.001 M-EDTA. After centrifugation for 2.5 h at 39,500 revs/min at 20°C in an IEC SB-283 rotor, O&ml fractions were collected from the bottom of the tube. 0.3-ml portions were taken for determination of acid-precipitable radioactivity and O.O5-ml samples were assayed for infectivity by transfection of E. coli K and K(P1) sphrroplast)s. The spheroplasts were prepared by the method of Henner et al. (1973) and the transfect,ion assay of Benzinger et al. (1971) was used. The sphoroplasts were prepared 18 to 24 h prior to their utilization. The efficiency of transfection (10e3 to 1O-4 plaque-forming units/h DNA molecule) was comparable to that obtained by Henner et al. (1973). E. coli K(P1) spheroplasts are able to restrict h DNA tha.t has not been modified in wivo by Pl methylation; i.e. X DNA has a relative transfection titer of 10d4 on E. coli K(P1) as compared to E. coli K spheroplasts. In contrast, h.(Pl) DNA transfects bot,h types of spheroplasts equally well. The degree to which K(P1) spheroplasts restrict X DNA transfection is similar in magnitude t,o the restriction of h phagc infection of intact K(P1) cells (Arber & Dussoix, 1962). (g) Puri&catiun

of the phage PI

restriction~motl~~cation

enzyme complex

The EcoPl enzyme complex was purified by the procedure of Brockes et al. (1972). Their procedure was followed through the phosphocellulose chromatography except for several modifications: (1) all buffers contained 0.58 m&I-PMSF, as suggested by Haberman (1974); (2) the crude extract was prepared by grinding the cells with twice their weight of alumina; (3) the methylase assay of Haberman et aZ. (1972), with the substitution of E. coli DNA as the substrate, was used to monitor enzyme activity. We found that Pl methylase activity (M*EcoPl) eluted from phosphocellulose in 0.1 fix-potassium phosphate, whereas Brockes et al. (1974) eluted the enzyme with 0.2 M-potassium phosphat,e buffer. One unit of Pl methylase act,ivity is defined as the amount of enzyme required t,o transfer 0.13 pmol of methyl groups from AdoMet to 2 pg of X DNA during 30 min incubation at 30°C. The enzyme was stored at -20°C in buffer containing 507; glycerol. Our enzyme preparation contained both Pl-specific methylase and endonuclease activities; under restriction (or methylation) assay conditions, h DNA (but not h. (Pl) DNA) was cleaved (or methylated). (h) In vitro

cleavage

by R.Ecol’l

Products of R .EcoPl cleavage were analyzed by sucrose gradient centrifugation. A X DNA (5.8 x lo4 cts/min reaction mixture (0.15 ml) containing 0.05 pg of 32P-labeled O,Ol M-2-mercaptoethanol, 0.006 per pg) in 0.1 M-TES (pH 7.7), 2 x 10m4 M-EDTA, M-Mgcl,, 0.02 M-acetic acid and 5x 10m4 M-ATP was used (Haberman, 1974.) 20 pg protein (0.05 ml) ofthe Pl enzyme preparation was added and the reaction incubated at 30°C for 1 h. The reaction was stopped by making up the mixture to 1.574 sodium dodecylsulfate and 0.02 M-EDTA, and heating to 65°C to disaggregate X cohesive ends. [3H]adenine-labeled h DNA was added as an internal marker. The finalvolumewas adjusted to 0.25 ml and the sample was layered onto a 4.4-ml linear sucrose gradient (lo?/, to 30%) in 0.02 M-Tris.HCl (pH 7.5), 0.02 M-NaCl, 0.001 M-EDTA. ARer centrifugation (2.5 h at 39,500 revs/min, 2O’C) in a Spinco SW50.1 rotor, O.15-ml fractions were collected and assayed for radioactivity. It should be noted that AdoMet was not included in the restriction reaction mixture. The R .EcoPl activity does not require AdoMet, although AdoMet does stimulate the rate of cleavage. However, in t,ho presence of AdoMet, thrre is compet)ition

‘I’ht> conditions for in ~:it~o methylation were the sanlc’ as tlcscri bed by Habcrman ct rzl. (1972). The reaction mistjure (0.15 ml) c~ontlainccl: 0.1 U-PIPES (pH6.8). 0.01 >I2 x 1W4 M-ED'L'A, 2 pg h DKA, 5 : 10 -7 ar-[3HjAdoMet (10 (‘i “-lll~rc~apt,oethanol, tumol) and O.OB ml (20 g protein) of the Pl enzyme preparation. After incubation iit, 30°C for 5 h, 0.02 ml of mllaheled 0.002 M-AdoMet was added and the reaction terminated acid. The precipit,atc was collect,ed I]> t)y pr,lcipitat,ion of the DNA in 5O, trichloroacetic filtration onto Whatman GFjA glass fiber disks and washed w&h 12 ml of’ lo,, trichloro:twtic acitl. The disks were dried and placed in scint,illation vials. Subsrqnently, the DPiA \vas rc?le;tscd from the filters by incubation for 4 h at, 60°C wit,h NCS solubilizer. l’hc* r,lclio;rctivity of’ each sample was then measured in a Packard licluid scintjillation counter. Incl\wion of thv NCS-r&we of DNA gave approxirnatcbly Ml”,, higher valnw thim filtt*rs cw~mt tl(l dirwt,ly.

3. Results (a) Methylution

of phage h DNA

by the T2 dam+ a&

damh

methylases

Although it, had been demonstrated t,hat the T2 damh enzyme methylated twoto threefold more &es on non-glucosylated, unmethylated. hmCyt-containing DNA t,han did t,he ‘I’2 rlam+ enzyme, no difference in met,hylation levels was found wit,h hcterologous. C-containing DNAs (e.g. calf thymus and micrococcal DNAs) (Revel $ Hattman. 1971; Hehlmann & Hattman, 1972). To det)errnine whether an>’ diRerewes in methylation existed with other C!-containing DNA substrates WV taxtended this analysis to phage h DNA. X DXS was methylated itz vitro with either the T2 rlarrr+ or damh enzyme using [14C lL4doMet as t,he methyl donor. After 60 minutes of incubat,ion. additional enz~nw was added t,o the reactlion mixt’ures. As shown in Table 1, the T2 dam+ enzyme added approximatjelS 150 methyl groups, whereas the ‘11’2dnmh enzyme added approximateI>, 1600 methyl residues per h DNA molecule. In both cases. addition of more cnzytnc. TABLE 1 h BNA

methytation

by the T2 dam+ or dam’-erqlrret

t Standard T2 tlurrr reaction mixtures (0.125 ml) were prepawd (we Materials and Mtsthods). [14C]Aclo;\let (56 mCi/mmol) was used as the methyl donor: 2 pg of h DNA were included in csach reaction. 1% u&s of T2 drcrn” and/or 95 units of T2 churn+ eJJzyme \verc a~lded/rcaction (bot,h enzymes were at 14,000 units/mg). After 60 min incubation at 3O’C’, vnzymo supplements \YW(~ atldcd as indiratotl and the incubation continued an additional 60 min. To terminate the rrartiolls. 40 p~nol unlabtlrd Ado&t were added to each reaction mix and t~hcaIINA then prwipiiatpd t,> the addition of 3 ml of 5% cold trichloroacetic acid. $ The average number of L’W]CH, groups added//\ DNA molecule was calculated after correcting for parallel controls containing boiled enzyme. The control valucx corresponded to only I to 2 methyl groups/h DNA molecule. The numbers in parentheses rcprcwntS t,hc variation from t 111. ,avwagr vale obt,ainwl in 3 separate experiments with the same ~~rzyrn~ 1)wparatiun.

386

J. E. BROOKS

AND

S. HATTMAN

at 60 minutes did not appreciably increase the amount of h methylation. Moreover, in separate reactions, T2 dam+ enzyme was added to damh methylated DNA and vice versa. As seen in Table 1, the damh enzyme could further methylate dam +-treated h DNA; i.e. the methylation level rose from 150 to 1600 MeAde residues per h molecule (the same methylation value as the damh enzyme alone). These results are similar to those obtained with non-glucosylated T-even DNA substrates (Revel & Hattman, 1971; Hehlmann & Hattman, 1972). It should be noted that the h DNA substrates used in the above experiments already contain 150 to 200 MeAde residues per h DNA (Hattman, 1972; our unpublished observations). These methylated residues are produced by the host DNA adenine methylase controlled by the E. coli dam gene (Marinus & Morris, 1973). The Eco dam enzyme methylates adenine residues within the sequence G-A-T-C (Lacks & Greenberg, 1977; Hattman et al., 197%); this sequence can be recognized by both T2 dam+ and darn” (Hattman et al., 1978a; see section (e) below). In a separate experiment, [3H]adenine-labeled h.dam, DNA (contains no MeAde) was methylated with either T2 dam+ or dam” (using unlabeled AdoMet as the methyl donor). The DNA was deproteinized by phenol extraction, precipitated in 70% ethanol, acid hydrolyzed and the bases separated by paper chromatography (Hatt’man, 1970) to determine the [3H]MeAde/[3H]Ade ratio. Bpproximately 600 methyl residues were added per h DNA by T2 dam +, and 2600 by T2 damh. Using different T2 enzyme preparations, we have observed variation in the absolute number of methyl groups added to A DNA substrates by both enzymes. Nonetheless, there was consistently a greater than threefold difference in methylation levels between dam+ and danah-treated h DNA. Furthermore, h DNA that was exhaustively methylated by the dam + enzyme could be further methylated by the damh methylase; t,he converse was not true. In all subsequent st’udies to be described, the dam+ and damh enzyme preparations used had similar specific act)ivites, and equal amounts of enzyme units were used to methylate the various h acceptor DNAs. Experiments were also conducted to determine the relative methylation of the two strands of the X DNA molecule. Unlabeled X DNA was mixed with h [32P]DNA and then methylated by the dam+ or damh enzyme using [3H]AdoMet. After dialysis, the samples were alkali denatured and the strands were separated by electrophoresis on agarose gels. After elution from the gel, the amount, of 32P and 3H was determined for each strand. It is clear that for a given methylase, the 3H/32P ratio is very similar for both t,he 1 and r strands (Table 2). Therefore, we conclude that both dam+ and darn” methylation sites are equally distribut’ed on the 1and T strands of the h DNA molecule. (b) Transfection of E. coli spheroplasts by in vitro methylated h DNA T2 damh, but not dam+, methylation protects T2 gt DNA against Pl restriction in vivo (Hattman, 1970; Revel & Hattman, 1971). Phage h DNA is also subject to PI restriction/modification (Arber & Dussoix, 1962). Since we had observed that h DNA is a substrate for both T2 dam + and T2 damh enzymes (and is hypermethylated by the latter), h DNA was used to determine whether the damh methylase could protect a heterologous (cytosine-containing) DNA against Pl restriction. ;\ DNA was methylated in vitro by the T2 dam+ or damh enzyme using [14C]AdoMet as the methyl donor. Following sucrose gradient centrifugation, fractions were collected and assayed for infectivity as well as for acid-precipitable radioactivity. As shown

T2

darn

METHYLATION

TABLE

Relative methylation h phage DNA

OF

X DNA

2

level of the 1 and r strads by the T2 dam enzymes

T strand 3H cts/min ‘1’2 rltrm+ ‘IY drrm”

(expt (expt (expt

1) 2) I)

833 711 4011

(expt 2)

349 I

32P ctsjmin

“H rts/min

“HW’

4.75 5.9”

175 120 159 106

740 696

25.2 32.9

4109 3439

of

I strand 321’ &s/min

156 124 I A.5 I I3

a,, ,:12,

.4.74 5.6 I

24.9 30-4

40 pg of unlabeled h DNA and 6 pg of h [32P]DNA (1.25 x lo3 cts/min per pg) were methylatc~ti at 30°C for I h in a standard T2 methylase reaction mixture (0.625 ml) containing [3H]AtloMct (0.8 (‘i/mmol) and 500 units of T2 dam+ or da&’ methylasc. Thr reaction mixtures ww dialywti against ()+)I wTris.HCl (pH 8.0), 0.001 M-EDTA (TE buffer), conwntratetl twofold by thalysis against solirl Sephadex and then dialyzed further in TE buffer. 2 to 4 pg of the 3H-mothyJattvl IINA were denatured and the strands separated by the method of Hayward (1972): the DNA was tknaturc~tl by the addition of 0.1 vol. 1 N-Nash. After 5 min at room temperature. (I.1 vol. of‘ a solution of 0.05% bromophenol blue in 600/” sucrose was added to the sample and the mixtun* M as immediately layered onto cylindrical O.fiOA,agarose gels (O.Svm j 20 cm). The DNA st~randswc~r~~ xcparated by electrophoresis for 15 h at 2.5 V/cm (4°C) in Trisiphosphate buffer (0.036 >I-‘l’riw. 0.03 M-N~H,P~~, WOO1 M-EDTA, pH 7.7). The gels were stainetl in Tris/phosphate buffw van taming 1 pg cthidium bromide/ml. The DNA was visualized by transillumination with ultraviokt light and the regions containing the individual 1 and ? strands were exrised. The DNA was elutrtl from the gel by incubation overnight at 60°C with NCS. The samples were then cwmtwl in t IIt, I’acltwrtl liquid scintillation rounter and the ratio of aH to 32P dotcrminecl.

I-ractmn (a)

(b)

““mDer (cl

Id1

Jqro. I. Transfection of ti. coli spheroplasts by in vitro methylatedh DNA. h DNA was methylatwl and then subjected to sucrose gradient centrifugation as described in Materials and Methods. Control X and X. (Pl) DNA substrates were incubated in the standard methylase reaction mixture. hut without any enzyme additions. Fractions (0.8-ml) were collected from the 11.5ml gradients and assayed for radioactivity and transfection ability. --o--o---, Acid-precipitable 14C ctajmin: --a-•-, plaque-forming units on E’. coli K spheroplasts; --A-A-, plaque-forming units on E. coli K(P1) spheroplasts. (a) X DNA methylated by T2 da.n~” enzyme. (b) X DNA methylato(J hy T2 dam+ enzyme, (c) X DNA control, (d) X’(Pl) DNA control.

388

J. E. RROOKS

AND

S. HATTMAN

in Figure l(c), t’he efficiency of t,ransfection by untreat,ed h DNA was 1W4 on E. coli K(P1) spheroplasts relative to E. coli K spheroplasts. In contrast, /\. (Pl) DNA can transfect spheroplasts of both strains equally well (Fig, l(d)). When X DNA was methylated by the T2 dam+ enzyme. it was still restricted 1W4 on E. coli K(P1) (Fig. l(b)). However, following methylation of h DNA \vith T2 damh, the t,ransfection of 0.28 efficiency on E. toll; K(P1) increased 3000-fold up to a relabivc infectivity (Fig. l(a)). It can also be seen (Fig. l(a) and (b)) that the pea,ks of infectivit’yand acid-precipitable radioactivity were coincident; this indicates that, methylated h DNA was not fragmented by a contaminating nuclease(s) during the in vitro incubation. In the above experiment, t)he dumb enzyme added about t’hreefold more methyl groups per X DNA molecule than did t’he dam+ enzyme (575 per X DNA for damh; 207 per A DNA for dam+). In a separate experiment’, X DNA was methylated it, vitro by eit’her the dam+ or damh enzyme (using unlabeled AdoMet). The DNA was deproteinized by phenol extraction, extensively dialyzed, and assayed for t)ransfection ability. The dam” methylation increased the relative transfect,ion efficiency of h on K(P1) spheroplasts over 7000-fold, to a level of 0.72; however, there was no protection of h DNA by t#he dam+ enzyme. We conclude that methylation by the damh (but not dam+) enzyme prot,ected A DNA against restriction in E. coli K(P1) spheroplasts. (c) R .EcoPl

cleavage

of in vitro

methylated

h

DNA

Since the above assays measured only the efficiency of spheroplast transfection, it still remained unanswered as t’o whether T2 danzh methylation protects A DNA against the direct action of Pl restrict,ion enz.yme; i.e. dam” mediated protection could be due to an undefined secondarp mechanism operating in the spheroplasts. Therefore, to eliminate such a possibility, it was necessary to deWmine directly the susceptibility of T2-met,hylat#ed X DNA to cleavage by purified R *EcoPl 32.P-labeled X DNA was methylated in, vitro wit,h t,he T2 dam, + or T2 dam,h enzyme; the met)hylat,ed DNA was incubat,ed wit,h ReEcoPl and then analyzed by sucrose gradient, cent)rifugation. As seen in Figure 2(c) control, untreat)ed h DNA was degraded by R. EcoPl to fragments shorter than unit length. h DNA t,hat \vas met)hylated with T2 dam+ enzyme was cleaved by R *EcoPl to the same extent) as the control h DNA (Fig. 2(b)). In contrast, A DNA methylated by the T2 dam” enzyme \vas not cleaved by R. EcoPl (Fig. 2(a)). Similar results were observed in experiments where t)he R -EcoPl digest)s were analyzed by agarose gel electrophoresis (dat,a not’ shown). These results indicate that T2 dam”, but not, T2 dam + . methylation directly protected A DNA against cleavage by R *EcoPl. In the next section, it will be shown t,hat T2 dam” is capable of methylating M.EcoPl recognition sites: the T2 dam+ methylase appears t,o be unable t,o methylat)e, or has a reduced affinit.v for, such sites.

((1) Ma EcoPl

methylatios of T2 dam-methylated

h I)Nd

It was of great interest to determine whether the T2 dam + and/or damh met’hylation sites overlap with those of MeEcoPl. To test this, h DNA was first methylated with either the dam+ or damh enzyme; the DNA was isolated and used as a substrate for methylation with MeEcoPl and [3H]AdoMet (as t,he methyl donor). The number of methyl groups added per phage chromosome was then calculated (Table 3). Control X DNA (not methylated wit,h either T2 enzyme) accepted approximately 30 methyl

T2

dam

METHYLATION

OF

Fraction

X

number (b)

(0)

3x9

DNA

(cl

Fro. 2. IC~E’cot’l cleavage of in vitro methyl&d X [32P]DNA. I.0 pg of h [s”P]DNA (5.8 1,’ lo4 cts/min per pg) was incubated at 30°C in a standard T2 methylase reaction mixture (1.25 ml) containing unlabeled AdoMet and 1200 units of T2 dam+ or dad methylase (14,000 mlits/mg for each methylase). In a control reaction, the h [aZP]DNA was incubated in a reaction mixture without any- enzyme addition. After 1.5 h of incubation, the reactions were heated to 60°C for 8 min, extracted with phenol, and then dialyzed extensivley against 0.62 wTris.HCl (pH 8.0) buffer. 0.05 pg of X [saP]DNA from each reaction was used in an H.EcoPI restriction assay, 01 incubated in the reaction mix without added enzyme (as a control), then subjected to SUCTOW gradient centrifugation. Prior to centrifugation, 0.2 pg of [3H]adenine-labelecl X DNA (7.0 x IO4 rts/min per pg) was added to each gradient to serve as an internal marker. Aft,er centrifugation, -a--a---, -+ Ii.EcoPI: --c ~~--\ .,. , fractions were collected and assayed for radioactivity. no enzyme. (a) X DNA methylated by T2 da& enzyme, (b) X DNA methylated by T2 tlnn,’ enzyme, (c) h DNA control. The arrow in each panel indicates the position of the [aH]adeninl,. labelrtl h DNA marker in the sucrose gradient.

TABLE 3

M .EcoPl Suhstratc h h.(Pl) h methylatetl X mcthylatetl

methylation

DNA

[aH]CH,

with with

T2 dam + T2 clan”

of X

DNL4t

residues 29.6 1.7 27.7 1.8

per h moleculcf

(24.7-30.9) (l+l.!l) (25.7-304) (162~1)

t 200 p.g of h DNA were mrthylated at 30°C in a T2 rlnrrb methylasr reaction mixture (2.5 ml) containing unlabeled AdoMet (80 PM) and 1600 units of T2 tlan~ + or da& methylase (14,000 units/mg for each methylase). After 1 h incubation, an additional X00 unit increment of T2 dam + and dam” was made to the respective reaction mixtures and the incubation continued. After 2 h the methylated DNAs were deproteinized by phenol extraction, dialyzed extensively against TE buffer (0.01 M-Tris.HCl (pH 8.0), 0.001 &I-EDTA) and then concentrated against dry Sephadex. The DNAs were methylated again by a repetition of the above procedure. The concentration of DNA was determined spectrophotometrically. Then 2 pg of each DNA sample were used as a substrate in a standard M.EcoPl methylation assay (see Methods and Materials). X DNA extensively rnethylated by T2 dam + received 200 methyl groups/rnol(,cltlc: h DNA extensively methylated by T2 damh received 1600 methyl groups/molecule. $ The numbers in parenthcxrs give the rango of mcthylation valws calculat,cd from 3 separate experiments. II

390

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E. BROOKS

AND

(a) III

A. HATTMAN

(b) 0

I

It

(cl 0

III0

FIG. 3. Sensitivity of iu oitro methylated X DNS to R. UpnI and R. UpnII. 200 pg of h.dam, DNA were methylated in a T2 dam methylase reaction mixture (2.5 ml) containing 14,000 units of T2 dam+ (24,000 units/mg) or 8000 units of T2 da& (14,000 units/mg) enzyme and unlabeled AdoMet (80 pM) as the methyl donor. A control reaction mixture was incubated without any enzyme addition. The reactions were incubated for 2 h at 30°C and then extracted twice with phenol. The DNA was concentrated against solid Sephadex and dialyzed extensively against 0.01 M-Tris.HCl (pH SO), 0.001 lr.EDTA. The DNA was then methylated a second time, phenolextracted, and concentrated by a repetition of the abovc procedure. After extensive dialysis against 0.02 iv-Tris.HCl (pH SO), the concentration of the DNA was determined spectrophotometrically. Methylated h DNA was then incubated with R. DpnI or R. DpnII using a modification of the standard reaction procedure (Lacks C Greenberg, 1977). Reaction mixtures (0.05 ml) contained 0.05 ix-Tris.HCl (pH 7.6), 0.05 M-NaCl, 0.005 wMgCl,, 0.5 pg h DNA and 0.05 units of R. DpnI

T’

tlnm

METHYLATION

OF

I

DNA

39 I

groups f’ollowing M .E’coPl methylat,ion. This number compares favorably with t hc value obtained by Haberman rt a,Z., (1972) for tho ijl vitro m&,vlation of h DNA b>. t,h(b Pl enzyme. (The number is low, however, when compared t,o t)he 70 additional MeAde re:sidues acquired during growth of h phage on E. cnli strains harboring I’1 prophage (Hatt,man, 1972).) In cont’rast, in, vitro methvlation of X.(Pl) DNA b> M .EcoPl was negligible: only one or two methyl residut>s were added per h. (I’1 ) DSA molecule (which has already been methylat,ed irf civo by M.EcoPl). h DNA that, had been methylated by the T2 da~n,~(~nzync was no longer a substrate for M. E’coPl methylation: e.g. M+EcoPl added only one t,o two methyl groups to h .rlaw~, premethylated with T2 dnnz” (Table 3). We conclude t,hat 7’2 Danny clnzymca can methylate M .E’coPl recognition sites. h .&we, DNA was met’hvlat,ed by T2 (Ju))~+ t#o a louver level than by T2 danzh, and t,his DNA accept,c~d t’he full complement of’ mct,hyl group3 from M.EcoPl (Table 3). (~1) Srquence

specijicity

of T2 dam + and darn” mrth,ylatiorr

In a prcbvious st’udy. no qualitative difference could be found in the scquentc S~JPdk!it~7 of ‘1’2 dam+ UP~SUGT2 damh: both enzymes appeared t’o methylate th(a et aZ., 1978~). adeninc residue within t,he sequence 5’ . . . G-A-Py . . . 3’ (Hattman Howevtlr, sincr the amalysis was not quantitative. it was still possible that the T2 du )I1* and tlarr/,” enzymes differ in their ability to met hylate a certain subset of sites within t*he group of G-A-Py sequences. To approach this question, we first investigated the ability of T2 dawl+ and danhI’ t,o methylate t’he sequence 5’ . . . G-A-T-C . . . 3’. When the adenine residue is m&hylatetl (t)o produce N6-M(,Ade) bhis sequence is subject to cleavage by R .I$nI: when t,hr sequence is unmethylated it is cleaved by R. I@I I or R.J!boI (Lacks & Greenberg. 1977: Gelinas et al.. 1977). Moreover, G-A-T-C is the : sequence methylatcd by M.&o dam (Lacks $ Greenberg, 1977: Hat,tman et al.. 197%). X DNA from phagt h.tlam, (devoid of MeAde) was methylated b!r T2 dam+ or daruh and then tested for its susceptibilit#v to cleavage (or met)hylation) by various nucleaeea (or met,hylases). As shown in Figure 3(a) and (b), bot’h T2 dam+ and T2 dam” met hylation rendered h .tlam, DNA sensitive to R. BpnI and resistant to R. f)p,r~l I (or R. Mbol). III contrast) h.dam, DNA is susceptible to R. L)pI/II. but) resistant to R.&n1 unmethylatetl ckavage. Furt’hermore, M .Eco darn could no longer methylat e t)hese modified DN\‘ds (dat,a not shown). Thestl results indicate t,hat, T2 dam + and dawrh met’hylated G-A-T-C sequences on h.dam, DNA. lt is significant, however, that the T2 methylated DNAs differed in their subst’rat’e activit,y wit,h M.EcoPl ; viz. damh mcthylation complet)elp blocked subsequent, M. EcoPl meth,ylation, whereas dam +- methylat,ion resulted in only a small (30$‘,,) rrduct.ion in M.EcoPl met’hylat,ion (dat,a not shown). These result,s or R~UJJ~II. After incubation at 37°C for 2 h, the reactions were tcwninatecl by the addition of 0.02 ml of a solution of BOoj, sucrose, 0.025 wEDTA, and 125 pg bromophenol blue/ml. The mixtures MWE loaded onto 1.4% agarose gels (0.6 cm x 14 cm) in Tris/borate electrophoresis buffw (0.089 m-T&, 0.08 sr-boric acid, 0.0025 ar-EDTA (pH 8.5)). The DKA fragments wrrc’ rwolverl by elertrophoresis at 150 V for 15 min, then 80 V f or 4 h (room tompwature). The gels wwc stained by soaking in Tris/borate buffer rontaining 1 pg othidium bromide/ml. DNA was visualized by transillumination with ultraviolet light; the gels were photographed on Polaroid Type 1Oi film through a red filter. (a) X .tlan,, DNA methylatetl by T2 dnn~ + enzyme, (b) X.&m,, l)?r’A mc~thylated by T2 duna” mzymr, (c) cv)ntrol X.duw, DN14’. I, IX. D/MI; i1, R . 11pnIl : (I. 110 vnzyme.

392

J.

E.

BROOKS

AND

S. HATTMAN

indicate that, although the G-A-T-C sequences were similarly protected by both T2 dam+ and damh, only T2 damh methylated Pl sites. In a separate experiment, equal amounts of X-dam, DNA were partially methylated (with [3H]AdoMet) to the same level by T2 dam + and T2 damh. Following enzymatic digestion, the (methyL3H)-labeled dinucleotides were isolated and analyzed by paper electrophoresis according to Hattman et al. (1978a). We observed that the proportion of A*-C was much higher with damh than dam+ at an early stage of methylation (Table 4). This was observed in two independent experiments, as well as at several stages of partial methylation (data not shown). These results suggest that, relative to clamh, the dam+ enzyme methylates G-A-C sequences much less efficiently than G-A-T sequences. TABLE 4 Analysis of methylated dinucleotides ,from h. dam, IjNA partially modified tcith T2 dam+ or T2 damh

Enzyme

dam + dumb

3H cts/min G-A* 7920 6600

t

in dinucleotidej A*-C A*-T 20-30 650

6570 3830

t 100 pg of X.dam3 DNA were methylated in the T2 dam methylase reaction (0.625 ml) containing [3H]AdoMet (1.6 Ci/mmol) and T2 dam+ or damh enzyme (650 units at 24,000 units/mg or 450 units at 14,000 units/mg, respectively) and incubated at 30% for 5 min. Under these dam + and dam” partially methylated the X.&m, DNA to the same level. 0.05 ml conditions, of unlabeled AdoMet was added to each reaction and the DNA immediately extracted with phenol. The DNA was dialyzed extensively against distilled water. $ [Methyl-sH]-labeled DNA was degraded with pancreatic DNase and B. coli exonuclease 1 and the dinucleotides isolated by ion-exchange chromatography (Hattman et al., 1978a). Portions of the diuucleotides were analyzed by paper electrophoresis at pH 1.9 (Hattman et al., 1978a). The sequence specificity of the T2 methylases is such that the only dinucleotides produced are G-MeAde (G-A*), MeAde-T (A*-T) and MeAde-C (A*-C) (Hattman et al., 1978a). It should be noted that A*-C/A*-T is much less than unity. This has been observed even with DNA methylated to higher extents (unpublished data). In general, the recovery of A*-C from pancreatic DNase digests is low; we believe that this is due mainly to the cleavage specificity of the enzyme (van Ormondt & Hattman, 1976).

4. Discussion The studies reported in this paper have shown the following. (1) The T2 dam+ and damh enzymes differ in their ability to methylate X DNA; the T2 damh enzyme methylates h DNA to a higher extent than does the T2 dam + enzyme when similar amounts of enzyme (at the same specific activity) are added to the reaction mixtures. The respective T2 dam + ldamh methylation sites are equally distributed on the 1 and r strands. (2) In vitro methylation by T2 dam h, but not T2 dam+, was able to protect A DNA against restriction during transfection of E. coli (Pl) spheroplasts. (3) T2 damh methylation completely protects X DNA against in vitro R .EcoPl cleavage, while under the same conditions T2 dam+ methylation affords no protection. (4) Bot,h T2 dam + and T2 damh methylation protected h. dam, DNA against cleavage by R. BpnII (or R *MboI) and against methylation by M 1Eco dam (each of these enzymes recognizes G-A-T-C). In contrast, only T2 damh methylation protected X DNA against any further methylation by MeEcoPl. (5) Xvdam, DNA preparations, methylated partially to the

T2 dam

METHYLATION

OF h DNA

Xl3

same level with T2 dam+ and T2 damh, exhibited large differences in the ratio of G-A-C to G-A-T sequences containing MeAde. It should be noted here that the M.EcoPl site is 5’ . . . A-G-A-C-Py . , . 3’ (Hattman et al.: 197%). In a previous study, it was concluded that the T2 dam+ and damh enzymes both et al., 197%~). From thtb nrrthylatje the sequence 5’ . . . G-A-Py . . . 3’ (Hattman rcwllts reporkd here we propose that, although both enzymes map methylate t’his certain of the G-A-Py sites. One wyuence? they differ in t’heir ability to methylate group of sequences, containing the trimer G-A-T, is efficiently met,hylated by both enzymes. In contrast, the group of sites containing G-A-C (including the M .EcoPl site) is metjhylated with a much lower efficiency by T2 danb + rtalative to its methylatjion \,y T2 &wzh. Moreover, it is possible that the damh enzyme may also prefer sequences containing G-A-T to those sites with G-A-C. Tt should be not,ed that differential ability to methylate G-A-C-containing sequences met,hylat8ion levels. does not, completely explain the difference in the T2 dam+/damh I f’the ‘l’2 dam” enzyme were able to methylat)e all G-A-Py sequences on h DNA (Mb”,, (i-1-C content), and the dam+ enzyme could only methylat8r: G-8-T sequences, only a twofold difference in met’hylation level would be expecbed. Yet,, much largw tliffcrcnccs were observed (Table 1) when X DNA was methylated under identical conditions. However, we have recently observed t,hat with a much higher enzyme-tol)NA ratio. t,he T2 O?CJW/,+ methylation level can be made to approach t,hat of t,he tlrrnrh enzyme. an ext,ensive examination of various physical properties has so far failed Taoreveal any differences bet,ween t’he dam + and darn” enzymes (Masuwkar C! Hatt,man, manuscript in preparation). Further studies art: in progress. The results obtained with i7~ vitro met,hylat,ed X DNA arc analogous t,o thosr for it/ V~VO met,hylat,rd T2 gt DNA. For example, mat,ure phage 1’2 gt dam+ and ‘1’2 c/t dawrh DNA differ t*wo- to tTsogens. whereas more than half the T2 gt danzh are resistant (Klein. 1965; Molholt, 1967; Revel & Georgopoulos. 1969). Thus, it appears t,hat compart~tl to datnh. the dnm + enzyme methylates Pl-specific sequences on T2 gf DNA much ltw efficientl,v t’han G-A-T-C sequences. ‘I’ht, authors WV ir~tlchted to Karen Pratt, for the plnification of‘ the Pl rrstric1iorl-m nrotiificat,ion enzyme complex. WC are grateful to Dr S. Larks for his generous gift, ot purified R,. &XIII. This work was supported by a Public Health Services grant no. AT-10864 and a Research Career Development Awurd no. AI 28022 (to 8. H.). One of tht aut,hors (J. E. B.) was a pretlootoral trainee, supported in part, by a, Yuhlic Health Srr\Tiws Training Grant no. S-TOl-GM-06658.

REFERENCES Arber. W. & Dussoix, D. (1962). J. Mol. Biol. 5, 18-36. Benzingcr, R., Kleber, I. & Huskey, R. (1971). J. l’irol. 7, 646-650. Brockes, J. P., Brown, P. R. & Murray, K. (1972). &o&em. J. 127, l-~10. Brockes, J. P., Brown, P. R. & Murray, K. (1974). J. NoE. Sol. 88, 437-443. Brooks, J. E. (1977). Ph.D. Thesis, Universit)y of Rochester. Brooks, J. E. di Hattman, 8. (1973). V’irology, 55, 285-288. (telinas, R. E., Myers, P. A. & Roberts, R. J. (1977). J. Mol. Sol. 114, 169- 179.

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Georgopoulos, C. P. (1969). Ph.D. Thesis, Massachuwtts Institntt, of T~~chnolopy. Haberman, A. (1974). J. ;VoZ. Riol. 89, 543-563. Haberman, A., Hrywood, J. & Mrsclson, M. (1972). 1 ‘tw. Sat. Awl. Sci., C’.S’.d. 69, 3138-3141. Hattman, S. (1964). Virology, 24, 333-348. Hattman, S. (1970). J’irology, 42, 359-367. Hattman, S. (1972). J. Viral. 10, 356-361. Hattman, S., van Ormondt, H. & dc Waard, A. (197%). J. it1oZ. Bicl. 119, 361-376. Hattman, S., Brooks, J. E. & Masurekar, M. (1987b). J. JIoZ. BioZ. 126, 367-380. Hayward, G. S. (1972). l’irology, 49, 342-344. Hehlmann, R. & Hattman, 8. (1972). J. %‘oZ. RioZ. 67, 351-360. Henner, W. D., Kleber, I. di Benzinger, R. (1973). J. l~irol. 12, 741-747. Klein, A. (1965). 2. I’ererbungsl. 96, 34(i-363. Lacks, S. & Greenberg, B. (1977). J. l%loZ. RioZ. 114, 153-163. Lederberg, S. (1957). I’iroZogy, 3, 496-513. Lehman, I. R. & Pratt, E. A. (1960). J. Biol. Chem. 235, 3254-3259. Lowry, 0. H., Rosenbrough, N. J., Farr, A. L. &. Randall, R,. J. (1951). J. BioZ. Chem. 193, 265-275. Marinus, M. G. & Morris, N. R. (1973). J. Bacteriol. 114, 1143 -1150. Molholtj, B. (1967). Ph.D. Thesis, University of Indiana. Reuben, R. & Gefter, M. (1974). ./. Biol. Chem. 249, 3843.-38.50. Revel, H. R. (1967). Virology, 31, 688-701. Revel, H. R. & Georgopoulos, C. P. (1969). I-irology, 40, l-17. Revel, H. R. & Hattman, S. (1971). l’irology, 45, 484-495. Revel, H. R. & Luria, S. E. (1970). Any&u. Rel?. Tenet. 4, 177-192. Revel, H. R., Hat,tman, 8. & Luria, S. E. (1965). Biuct~em. Biophy~. Res. Commun. 18, 545-550. Schallcr, H., Nusslein, C., Bonhoeffcr, F. J., Kurz, C. 8r. Nictzsrhmann, I. (1972). E’ur. ,J. Biochem. 26, 474-481. Smith, H. 0. & Nathans, D. (1973). J. ~1102. BioZ. 81, 419 ~423. van Ormondt, H. & Hattman, S. (1976). Anal. Biochem. 74, 207-213. Wyatt, G. R. & Cohen, S. S. (1952). Nature (London), 170, 1072-1073. Yamamoto, K., Alberts, B., Benzinger, R., Lawhornc, L. & Trribcr, G. (1970). JTiroZogy 40, 734-744.