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Biochimica et Biophysica Acta, 562 (1979) 418--428
© Elsevier/North-Holland Biomedical Press
BBA 99445
THE E F F E C T OF D I F F E R E N T I A L METHYLATION BY E S C H E R I C H I A C O L I OF PLASMID DNA AND PHAGE T7 AND k DNA ON THE CLEAVAGE BY RESTRICTION ENDONUCLEASE M b o I FROM M O R A X E L L A B O V I S
BRIGITTE DREISEIKELMANN, RUDOLF EICHENLAUB and WILFRIED WACKERNAGEL
Lehrstuhl Biologie der Mikroorganismen, Ruhr-Universitf~'t, D-4630 Bochum (F.R.G.) (Received September 1st, 1978)
Key words: Methylation; Plasmid DNA, Restriction endonuclease; (E. coli mutant, Phage)
S u m m a r y
The nucleotide sequence recognized and cleaved by the restriction endo- nuclease M b o I is 5'~GATC and is identical to the central tetranucleotide of the restriction sites of B a m H I and BglII. Experiments on the restriction of DNA from E s c h e r i c h i a coli d a m and d a m ÷ confirm the n o t i o n t h a t GATC sequences are adenosyl-methylated by the d a m function of E. coli and t h e r e b y are made refractory to cleavage by M b o I . On the basis of this observation the degree o f d a m m e t h y l a t i o n of various DNAs was examined by cleavage with M b o I and o t h e r restriction endonucleases. In plasmid DNA essentially all of the GATC sequences are m e t h y l a t e d by the d a m function. The DNA of phage ~ is only partially m e t h y l a t e d , extended m e t h y l a t i o n is observed in the DNA o f a sub- stitution m u t a n t of ~, ~ galsbio2s6, and in the ~ derived plasmid, ~dv93, which is completely m e t h y l a t e d . In contrast, phage T7 DNA is n o t m e t h y l a t e d by dam. A suppression of darn m e t h y l a t i o n of T7 DNA appears to act only in cis since plasmid DNA replicated in a T7-infected cell is completely m e t h y l a t e d . The results are discussed with respect to the participation of the darn methyl- ase in different replication systems.
I n t r o d u c t i o n
The restriction endonuclease M b o I isolated from M o r a x e l l a bovis recognizes 5'~ GATC 3'
the nucleotide sequence 3' CTAG~5' and cleaves at the sites marked by the arrows [1]. The e n z y m e produces 'sticky ends' o f the sequence GATC at the
cleavage site and thus should be suitable for molecular cloning of DNA frag- ments. The sequence GATC represents the central tetranucleotide o f the recog- nition sequence of other restriction endonucleases, among them the enzymes BamHI isolated from Bacillus amyloliquefaciens [2] and BgIII isolated from 5'C#GATC G3' and BglII the Bacillus globigii [3]. BamHI cleaves the sequence 3'G CTAGtC5'
5'A ~ GATC T3'
sequence 3'T C T A G t A 5 ' at the sites indicated b y arrows. Therefore, MboI is expected to cut any BamHI and BgIII site and thus l~as been proposed to be useful for cloning o f MboI-derived restriction fragments into BamHI and BglII sites of appropriate vehicles [ 1,4]. However, when we compared the restriction pattern of MboI, BamHI and BglII with the DNA o f the composite plasmid pFE42, we observed instead of at least six cuts with MboI (three BamHI sites and three BgIII sites) barely one in the plasmid DNA. Since this result suggested that modification of the DNA by methylation was involved, we examined the restriction of plasmid and phage DNAs by MboI and other restriction endo- nucleases with respect to DNA methylation conferred b y the host cell, in particular by the d e o x y a d e n o s i n e methylase coded b y the dam gene [5]. The results indicate that in plasmid DNA the MboI restriction sites are m e t h y l a t e d b y the dam function and t h e r e b y made refractory to cleavage. In phage DNA the adenosyl-methylation o f GATC sequences appears to vary depending on the respective phage and also on its particular genetic constitution. The results are discussed with respect to the participation o f the dam methylase in different replication systems.
Materials and Methods
Escherichia coli strains. Plasmids were generally maintained and isolated from C600 (thr, leu, thi, tonA). Other strains were SF8 (recB21, recC22, lop-11, tonAl, thr-1, leu-6, thi-1, lacY1, supE44, rKmK), A B l 1 5 7 dam-4 (argE3, his-4, thr-1, leu-6, proA2, thi-1, strA31, lacY1, gal, ara, xyl, mtl, strA, from B. Glickman) and WA 834 (met r~m~; from W. Arber).
Isolation o f plasmid DNA. The plasmids p F E 4 2 and p B R 3 2 2 as all ColE1- containing chimeras can be amplified b y chloramphenicol [6]. A 500 ml cul- ture o f a plasmid-containing strain was aerated in t r y p t o n e - y e a s t extract m e d i u m at 37°C. At a titer of 3 • 108/ml chloramphenicol (Sigma) was added to a final concentration of 250 pg/ml. The culture was then aerated for another 16 h at 37°C. A Triton-lysate was prepared as described [7] with some small modifications. The cells were harvested b y centrifugation and resuspended in 8 ml TES (100 mM Tris, p H 7.5, 50 mM NaC1, 5 mM EDTA) containing 25% sucrose. Spheroplasts were prepared b y addition of 2 ml of l y s o z y m e (Serva, 10 mg/ml) and 4 ml o f 0.25 M EDTA. After 15--20 min at 0°C the spheroplasts were lysed b y addition of the same volume of 'lytic mix' consisting o f 0.4% Triton X-100 (Serva) in 50 mM Tris-HC1, pH 7.5, 62.5 mM EDTA [8]. After 20 min at 0°C the lysate was centrifuged for 20 min at 35 000 X g. The supernatant was subjected to CsCl-gradient centrifugation in the presence of ethidium-bromide as described [8]. The supercoiled D N A was collected from the gradients and the centrifugation was repeated. Ethidium
420
b r o m i d e was r e m o v e d by e x t r a c t i o n three times with isopropanol saturated with CsC1. The plasmid DNA was t h e n dialysed against 10 mM Tris-ttC1, pH 7.5, 1 mM EDTA. DNA o f the plasmid Xdv93 was a gift o f M. Lusky from t h e l a b o r a t o r y o f Dr. G. Flobom.
Isolation o f phage DNA. DNA from a T7 + strain (F.W. St/tdi~r)was isolated according to Seroka and Wackernagel [9]. X DNA was isolated after phenol e x t r a c t i o n o f phage purified by CsC1 gradient centrifugation. Xci857 Sam7 was prepared b y t h e r m o - i n d u c t i o n o f E. coli strains lysogenic for this phage. ), cI857galsbio2s6 [10,11] was o b t a i n e d after infection o f cells in liquid culture.
Restriction endonucleases. MboI was isolated according to Gelinas et al. [1], BglII according to Pirrotta [3], EcoRI according to Greene et al. [12], [tmdII/
III according to Smith and Wilcox [13], and Sau3AI according to Sussenbach et al. [14]. BamHI was purchased from Miles Lab. (Elkhart, IN, U.S.A.) and PstI and KpnI were from Biolabs (Beverly, MA, U.S.A.). In some e x p e r i m e n t s an MboI p r e p a r a t i o n from Biolabs was also used.
DNA cleavage by restriction enclonucleases. Reaction conditions for restric- tion with EcoRI were 100 mM Tris-tlC1, pH 7.5, 50 mM NaC1, and 10 mM MgC12. R e a c t i o n c o n d i t i o n s for BamHI, BglII, KpnI, mad MboI were 6 mM Tris-HC1, p i t 7.5, 6 mM MgC12, and 6 mM ~-mercaptoethanol. In some cases 150 mM NaC1 plus bovine serum albumine (100 ~g/ml) were included in reac- tions with Mbol. Reaction mixtures for HindII/III c o n t a i n e d 6 mM Tris-ltCt, pH 7.5, 6 mM MgC12, 1 mM d i t h i o t h r e i t o l and gelatine (0.1 mg/ml). Iteaclion m i x t u r e s were incubated at 37°C for a p p r o p r i a t e times, mostly two to three h.
Reaction conditions for Sau3Al were 6 mM Tris-HC1, pll 7.5, 15 mM MgCI~, 60 mM NaC1, and 6 mM ~-mercaptoethanol. The incubation was at 3 0 C .
Electrophoresis. Agarose gel electrophoresis was p e r f o r m e d as described [12] using 5-mm slab gels (16 : 11 cm) and run at 100 V and 40 mA for al) a p p r o p r i a t e time, generally a b o u t 2.5 h. P o l y a c r y l a m i d e gels (4%, 16 11 cm}
o f 1.5 m m width were prepared according to Loening [15] and run at 100 V and 40 mA for 5 h in a Tris-Phosphate b u f f e r [16].
Visualization o f DNA fragments. Gels were stained with ethidium b r o m i d e and p h o t o g r a p h e d on a C h r o m a t o - V u e transilluminator, m o d e l C-62 (Ultra- Violet Products Inc., San Gabriel, CA, U.S.A.). Photographs were taken on Polaroid Land film t y p e 665 or on Ilford film Pan F 50ASA.
Transformation. T r a n s f o r m a t i o n was carried o u t as previously described [17] e x c e p t that a c o n c e n t r a t i o n o f 80 mM CaC12 was used. Plasmid DNA ( b e t w e e n 0.5 and 2/Lg) was diluted in 10 mM Tris-HC1, pH 7.5, to a volume o f 0.1 ml. The t r a n s f o r m a n t s were selected for their resistance to penicillin by plating samples o f the t r a n s f o r m a t i o n m i x t u r e on c o m p l e t e agar which con- tained 200 gg/ml penicillin (Serva).
Results
1. Restriction o f pFE42 DNA
Plasmid p F E 4 2 ( 1 3 . 3 . 106 daltons; Eichenlaub, R., unpublished data) is c o m p o s e d o f R S F 2 1 2 4 (ColE1 carrying TnA) [18] and mini-F [8]. This plasmid contains three BamHI and t h r e e BglII restriction sites (Fig. 1; slots C and D). When p F E 4 2 DNA was digested with MboI, only one cut was intro-
duced into a fraction of the DNA molecules (Fig. 1 ; slot E), even with excess of enzyme. This was surprising, since at least six cuts per molecule were expected corresponding to the BamHI and the BglII sites. In fact, significantly more than six MboI sites might reside in pFE42 DNA due to the smaller recognition sequence of MboI compared to BamHI and BgIII (see introduction). In order to verify t h a t our preparation of MboI indeed displayed the published cleavage specificity DNA of phage T7 was also restricted with MboI (Fig. 1 ; slot A). The restriction pattern obtained was identical to t h a t reported by McDonell et al.
[19] except t h a t the two small fragments of 408 and 120 base pairs are n o t visible on the gel shown in Fig. 1.
Although only a fraction of the pFE42 DNA molecules was linearized by MboI we a t t e m p t e d to localize the MboI cleavage site on the plasmid DNA. For this purpose double restriction with MboI plus KpnI was performed. Since this did n o t produce one specific extra fragment besides the two KpnI fragments it was concluded t h a t the sporadic MboI cleavages cannot be attributed to one specific site on the DNA. However, d o u b l y restricted DNA produced a set o f discrete bands of smaller molecular weights on the gel (Fig. 2; slot B). This observation argues against a contaminating unspecific endonuclease in our
. . . . i ... i ~ ' ~
... = ~ ... = ! !!!~!i i?iii~i!!!~ ii?i~ii!!i~i!!~ ~i~ ~7~ ¸ ~
A B C : D : : E
F i g . 1. A g a r o s e g e l e l e c t r o p h o r e s i s o f T 7 D N A a n d p F E 4 2 D N A t r e a t e d w i t h v a r i o u s r e s t r i c t i o n e n d o - n u c l e a s e s . A , T 7 D N A p l u s M b o I ; B, p F E 4 2 D N A , n o e n z y m e ; C, p F E 4 2 D N A P l u s B a m H I ; D, p F E 4 2 D N A p l u s BglII; E, p F E 4 2 D N A p l u s M b o l . T h e a g a r o s e c o n c e n t r a t i o n w a s 0 . 8 % .
F i g . 2. A g a r o s e gel e l e c t r o p h o r e s i s o f p F E 4 2 D N A t r e a t e d w i t h M b o I ( A ) and w i t h M b o I p l u s K p n l ( B ) . D N A t r e a t e d w i t h E e o R I ( C ) s e r v e d as r e f e r e n c e f o r t h e m o l e c u l a r w e i g h t o f t h e f r a g m e n t s . T h e a g a r o s e c o n c e n t r a t i o n w a s 1 . 0 % .
4 2 2
preparation of MboI, a notion which is further s u p p o r t e d b y the ability to ligate the plasmid DNA linearized by MboI (Dreiseikelmann, B., unpublished results). The same results were obtained with a commercial preparation of MboI. Thus, the p r o d u c t i o n of a series of discrete fragments u p o n double restriction with MboI plus KpnI is consistent with the assumption that MboI cleaves p F E 4 2 DNA occasionally at one of several sites producing a population o f discontinuously p e r m u t e d linear molecules.
The fact that MboI cuts the plasmid p F E 4 2 containing three BamHI and three BglII sites barely once suggested that DNA methylation by the host cell m a y play a role in the restriction of DNA by MboI. An influence of the host specific modification system was excluded b y the observation that p F E 4 2 DNA isolated from E. coli SF8 rKm K and E. coli m e t rBm ~ was as refractive as wild t y p e DNA to restriction by MboI. However, when p F E 4 2 DNA was isolated from E. coli dam-4 which is defective for the major adenine methylase (Ref. 5; Glickman, B., personal communication), the restriction pattern for MboI was dramatically changed (Fig. 3; slot C). MboI n o w cleaved p F E 4 2 DNA into more than t w e n t y fragments.
+ + - + - + - +
A B C D E
Fig. 3. A g a r o s e gel e l e c t r o p h o r e s i s o f D N A r e p l i c a t e d in E. c o l i w i l d t y p e (+) a n d E. c o l i darn ( - - ) a n d t r e a t e d w i t h v a r i o u s r e s t r i c t i o n e n d o n u c l e a s e s . A, T 7 D N A p l u s M b o I ; B, p F E 4 2 D N A , n o e n z y r n e ; C, p F E 4 2 D N A p l u s M b o I ; D, p F E 4 2 D N A p l u s B a r n H I ; E, k D N A p l u s H i n d I I / n I . T h e a g a r o s e c o n c e n t r a - t i o n i n t h e gel w a s 0.8%.
Furthermore it was observed that the sporadic cleavage o f d a m - m e t h y l a t e d plasmid DNA b y the restriction endonuclease M b o I is depending on the salt concentration. The linearization of p F E 4 2 DNA b y M b o I is abolished in the presence o f 100 mM NaC1, a condition which does n o t interfere with the cleavage of T7 DNA. Therefore it seems that the few cuts p r o d u c e d b y M b o I in m e t h y l a t e d DNA cannot be explained by the incomplete methylation o f GATC sequences b u t may rather be due to some intrinsic activity of M b o I which occasionally cleaves the m e t h y l a t e d sequence at low salt concentration.
These results support the notion made on the basis of experiments with M b o I and other restriction endonucleases [1,3,20] that M b o I favourably cleaves u n m e t h y l a t e d recognition sequences and that the dam gene p r o d u c t modifies all M b o I sites in plasmid DNA.
2. Restriction o f k D N A
M b o I cleaves k DNA into more than 50 fragments [1]. We compared the cleavage of k DNA isolated from E. coli darn + b y M b o I and S a u 3 A I , a restric- tion endonuclease from Staphylococcus aureus which also recognizes and cleaves the nucleotide sequence 5'*GATC [14]. Even with an excess of M b o I only a partial fragmentation of k DNA was obtained in darn + experiments whereas S a u 3 A I p r o d u c e d more than 50 fragments (Fig. 4; slots A + and B) in
÷ - ÷ .... . . . . + - .. . .
A C D • E
Fig. 4 . P o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s o f b a c t e r i o p h a g e k D N A r e p l i c a t e d in E. coli w i l d t y p e (+) a n d E. c o i l d a m ( - - ) t r e a t e d w i t h r e s t r i c t i o n e n d o n u c l e a s e s M b o I a n d S a u 3 A I . A , k + D N A p l u s M b o I ; B , k+
D N A p l u s S a u 3 A I ; C, T 7 D N A p l u s H i n d I I / I I I ; D, k g a | s b i o 2 5 6 D N A p l u s M b o I ; E, k g a l s b i o 2 S 6 D N A p l u s S a u 3 A I . P o l y a c r y l a m i d e c o n c e n t r a t i o n w a s 4%, M b o I d i g e s t s w e r e w i t h 1 5 0 m M NaCI.
424
either d a m + or d a m experiments. With )~ DNA from a d a m strain these frag- ments were also p r o d u c e d by M b o I (Fig. 4; slots A- and B-). These data are consistent with the finding b y fingerprint analysis of cohesive ends with the sequence GATC that in ~ DNA the adenine in GATC is only approx. 50%
m e t h y l a t e d [3]. Thus, adenine-methylated GATC sequences are probably not cleaved b y M b o I , b u t S a u 3 A I cleaves GATC irrespective of methylation.
Interestingly, the DNA of a plasmid derived from ~, kdv93, which consists of a 4.7% fragment of ~ ranging from 78.2% to 82.9% o f the total length of DNA [21], is not cleaved by M b o I indicating essentially complete methylation of GATC sequences (Fig. 5). When we tested the m e t h y l a t i o n o f GATC in the DNA o f a substition m u t a n t of ~, ~galsbio2s6 ci857, b y restriction with M b o I and S a u 3 A I , the DNA appeared to be almost completely m e t h y l a t e d (Fig. 4; slots D and E).
3. R e s t r i c t i o n o f T 7 D N A
The DNA of T7 is cleaved by M b o I into seven fragments [19]. T7 DNA replicated in a d a m + and a darn strain of E. coli gave identical restriction patterns with M b o I which are indistinguishable from restriction patterns obtained with S a u 3 A I (data n o t shown). Thus, ill T7 DNA the GATC sites are
Fig. 5. P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s o f k d v 9 3 D N A f r o m a n E . c o l i d a r n + s t r a i n t r e a t e d w i t h restric- t i o n e n d o n u c l e a s e s M b o I and S a u 3 A I . A, k d v 9 3 D N A p l u s M b o I ; B, k d v 9 3 D N A plus S a u 3 A I (7 D N A b a n d s ) . P o l y a c r y l a m i d e c o n c e n t r a t i o n w a s 6.3%.
presumably n o t m e t h y l a t e d b y the host cell. The mechanism which prevents dam m e t h y l a t i o n of T7 D N A could be analogous or identical to the process which suppresses host specific modification o f T7 D N A and which involves the function of gene 0.3 [22,23]. However, DNA of a deletion m u t a n t of T7, D104 am 193LG3, in which one deletion extends over the c o m p l e t e gene 0,3 [24], is still devoid of m e t h y l a t e d GATC sequences as evidence b y the unimpaired cleavage b y MboI (data n o t shown). Nevertheless, a suppressive mechanism controlled b y genes not deleted in the T7 m u t a n t could still be operative. It has recently been d e m o n s t r a t e d by conjugation experiments that infection o f a cell receiving an F ' f a c t o r b y ultraviolet-inactivated T7 results in a suppression of the host specific restriction of the F ' D N A [25]. Therefore, a trans-test for possible suppression of dam* activity was performed. Growing cells o f E. coli W3550 su- carrying the plasmid p B R 3 2 2 were infected at a multiplicity o f 10 with T7 a m 2 8 a m 2 9 a m 2 3 3 defective for DNA polymerase, endonuclease and exonuclease [ 26]. The phage was irradiated with ultraviolet light prior to infec- tion to give a survival of 10-s; 15 min after infection at 37°C (control: uninfected
A e
Fig. 6. P o l y a c r y l a m i d e gel electrophoresLs of plasmid p B R 3 2 2 DNA r e p l i c a t e d in E. coli wild t y p e a n d in E. coli wild t y p e a f t e r i n f e c t i o n w i t h u l t r a v i o l e t - i n a c t i v a t e d T 7 a m 2 8 a m 2 9 a m 2 3 3 t r e a t e d w i t h r e s t r i c t i o n e n d o n u c l e a s e s M b o l a n d S a u 3 A I . A, p B R 3 2 2 DNA from T7 infected E. eoli d a m + Plus M b o I ; B, p B R 3 2 2 DNA from n o r m a l E. coli dam+; C, p B R 3 2 2 DNA from T7 infected E. coli d a m + plus S a u 3 A I ; D, p B R 3 2 2 DNA f r o m n o r m a l E. coli darn + plus S a u 3 A I . P o l y a c r y l a m i d e c o n c e n t r a t i o n w a s 6.3%.
426
cells) chloramphenicol at 250 ~g/ml was added to the culture in order to block f u r t h e r protein synthesis and one h o u r after infection [3H]thymidine was added. Aeration was c o n t i n u e d for 14 h to amplify the plasmid. Supercoiled pBR322 DNA was then isolated. The radioactivity in this DNA showed t h a t plasmid DNA had replicated in the infected cells. A comparison of label incor- porated into pBR322 supercoils in infected and uninfected cells (control) indicated t h a t at least 80% of the supercoil DNA in infected cells was newly synthesized. The DNA was digested with M b o I and Sau3AI. As shown in Fig. 6 (slots A and E) pBR322 DNA from the T7 infected cells was as refractive to cleavage by M b o I as DNA from u n i n f e c t e d cells, whereas S a u 3 A I cleaved both plasmid DNAs into discrete fragments (Fig. 6; slots C and D). This result shows t h a t the plasmid DNA is still normally d a m - m e t h y l a t e d after replication in a T7 infected cell. It appears t h a t in contrast to modification-specific m e t h y l a t i o n a suppression of the dam methylase activity does n o t occur.
Discussion
The restriction endonuclease M b o I which recognizes the sequence 5'GATC [1] cleaves DNA only when it is n o t m e t h y l a t e d by the dam function of E. coli. This interpretation of our results implies t h a t GATC is one of the sequences which are recognized by the dam e n z y m e and which are specifically m e t h y l a t e d at the internal adenine. This conclusion is in accord with the observation by Gelinas et al. [1] t h a t adenovirus-2 DNA is a better substrate for M b o I t h a n DNA from }~ grown in E. coli K12. It is also c o n s o n a n t with the finding by Pirrotta [3] t h a t the adenine residues in GATC sequences in ~ DNA are partially m e t h y l a t e d by the dam function of E. coli. Recently Lacks and Greenberg [20] have made a similar s t a t e m e n t on the basis of their finding t h a t the restriction endonuclease D p n I I from Diplococcus p n e u m o n i a e cleaves the nucleotide sequence GATC when it is n o t m e t h y l a t e d by the dam function.
Thus, M b o I and D p n I I are identical in cleavage specificity with respect to recognition sequence and m e t h y l a t i o n [20,27]. On the other hand, the enzymes BarnHI [2] and BglII [3] having GATC as the central tetranucleotide in their hexanucleotide recognition sequences cleave plasmid DNA irrespective o f dam m e t h y l a t i o n (Fig. 1), a result that supports earlier observations obtained with partially m e t h y l a t e d DNA [3,28].
Our results with plasmids pFE42, pBR322 and ~dv93 show t h a t in plasmid DNA essentially all GATC sites are normally m e t h y l a t e d by the dam function in E. coli K12 and t h a t an enzyme with identical specificity exsists in E. coli B.
The DNA of phage ~ wild type appears to be partially m e t h y l a t e d by dam, a result which is in accord with published data [3,28]. The almost complete m e t h y l a t i o n of GATC sequences in h galsbio2s6 DNA remains unexplained b u t it seems n o t unreasonable to assume that a gene in ~, lost through the galsbio256 substitutions and also absent in hdv93, controls the e x t e n t of dam m e t h y l a t i o n by the host cell. F u r t h e r studies will be directed to attribute this control f u n c t i o n to a specific gene of phage h.
The total lack of GATC m e t h y l a t i o n in T7 DNA contrasts with t h e partial or complete m e t h y l a t i o n of ~ DNA and plasmid DNA, respectively. By coin- cidence, this order in m e t h y l a t i o n density m a y be correlated to the mode of
DNA replication in that plasmid DNA replicates only as a circular molecule, DNA replicates as a circle early and as a linear molecule late during intra- cellular development [29,30] and T7 D N A replicates exclusively via linear intermediates [31--33]. However, a correlation appears n o t very plausible if one considers that the individual nucleotide sequences recognized b y a transfer- ase should n o t differ in a circular and a linear DNA molecule. One could also argue that phage DNA may n o t allow for extensive methylation before it is packaged into phage particles. However, the observed difference in m e t h y l a t i o n density between T7 and k and even b e t w e e n different k variants cannot be explained easily. An alternate interpretation relates dam methylation to the t y p e o f replication system that is active in the replication of the different DNAs. The plasmid DNA in our experiments, either ColE1, mini-F or kdv is replicated by the enzymes which also replicate the chromosomal DNA. The virulent bacteriophage T7, on the other hand, provides its own replication apparatus. An intermediate case is the temperate phage k which depends largely on host functions for replication. Our tentative interpretation would then be that the dam methylase is an integral c o m p o n e n t o f the E. coli replication system so that DNA replicated b y this system is c o n c o m i t a n t l y being dam- methylated. The T7 replication apparatus apparently does n o t require or allow the participation o f the dam product. This notion is in agreement with our observation that plasmid DNA is being normally dam-methylated when repli- cating in a T7-infected cell. A necessity of the dam function for normal E. coli replication is evident from the manyfold physiological alterations observed in dam~leficient mutants of E. coli, such as increased spontaneous mutability, filamentation, high spontaneous induction of prophage k, accumulation of single-strand interruptions in chromosomal DNA, hyper-recombination pheno- t y p e and inviability o f dam double mutants with polA and recA, recB or recC [34,35].
Acknowledgements
We thank Dr. R.J. Roberts for providing experimental data prior to publica- tion and a sample o f Moraxella bovis and Dr. B. Glickman for the dam-4 m u t a n t of E. coli. Thanks are also due to Dr. W. Rtiger for generous gifts o f restriction endonuclease E c o R I and HindII/III. We are also indepted to Dr. G.
H o b o m for his valued advice and his gift of kdv93 DNA. The expert technical assistance of Brigitte Thoms during some of the experiments is gratefully acknowledged. This w o r k was supported by the Deutsche Forschungs- gemeinschaft.
References
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