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Proc. Natl. Acad. Sci. USAVol. 83, pp. 9070-9074, December
1986Genetics
Escherichia coli K-12 restricts DNA containing
5-methylcytosine(,rg1/rgB/molecular cloning)
ELISABETH A. RALEIGH AND GEOFFREY WILSONNew England Biolabs,
Beverly, MA 01915
Communicated by Allan M. Campbell, July 21, 1986
ABSTRACT We have observed that plasmids containingcertain cloned
modification methylase genes of type H restric-tion-modification
systems cannot be transformed into manylaboratory strains of
Escherichia coli K-12. The investigationof this phenomenon,
reported here, has revealed (i) DNAcontaining 5-methylcytosine is
biologically restricted by thesestrains, while DNA containing
6-methyladenine is not; (ii)restriction is due to two genetically
distinct systems that differin their sequence specificities, which
we have named mcrA andmcrB (for moiffied cytosine restriction).
Since 5-methylcyto-sine containing DNA is widespread in nature, the
Mcr systemsprobably have a broad biological role. Mcr restriction
mayseriously interfere with molecular cloning of
5-methylcytosine-containing foreign DNAs. The Mcr phenotypes of
some com-monly used strains of E. coli K-12 are reported.
When foreign DNA is introduced into bacteria, it is frequent-ly
attacked by restriction systems encoded by the host cell(1-4). An
important feature of the action of these systems isthat the
survival of a DNA molecule in a restricting hostdepends not only on
its sequence but also on its history: thesame sequence behaves
differently depending on what its lasthost was. The particular host
in which the molecule replicatesconfers on the DNA a modification,
usually methylation ofanadenine or cytosine residue within the
target sequence, thatprotects it against the cognate restriction
functions; it is notprotected against heterologous restriction. It
is generallyassumed that restriction provides a defense against
invasionby foreign DNA, especially phage DNA, and that
cognatemodification serves primarily to prevent suicidal attack
onhost DNA.The first restriction systems to be described were the
Rgl
(for restricts glucose-less phage) systems (1, 5, 6). Thesewere
identified as functions that attack T-even phages, butonly when
they contain 5-hydroxymethylcytosine in theirDNA. The T-even phages
incorporate 5-hydroxymethylcy-tosine intoDNA at the mononucleotide
level; glucosyl groupsare later transferred to
5-hydroxymethylcytosine in thepolynucleotide from UDP-glucose by
phage-encoded en-zymes. The phage DNA is sensitive to the RglA and
Rg1Brestriction functions only when the 5-hydroxymethylcytosineis
not glucosylated, as when the glucosyltransferase enzymesare
defective, or when the host lacks UDP-glucose. Mutantsof T4 that
contain completely unmodified cytosine are notrestricted.We
describe here two restriction systems present in Esch-
erichia coli K-12 that attack DNA containing methylatedcytosine
in particular sequences. In the course of cloning themethylase
genes associated with restriction-modificationsystems, we
encountered difficulty in transforming certainclones into many
common E. coli K-12 strains. We foundexisting variation among
laboratory strains for this methyl-ase-rejection property, and we
have begun a genetic study of
the phenomenon. This rejection has the formal properties
ofhost-controlled restriction systems. It is probably identical
toRgl restriction of nonglucosylated T-even phages (E.A.R.,
R.Trimarchi, and H. Revel, unpublished observations; also
seebelow).
MATERIALS AND METHODSBacterial and Phage Strains. Strains were
obtained from the
following sources: W3110, C600, and CR63 (7), N. Kleckneror N.
Murray; HB101, RR1, and LE392 (8), our collection orN. Kleckner;
K802 (9) and MM294 (10), our collection;JM107 (11) and JM107 MA2
(12), R. Blumenthal; JM101 (11),J. Messing; Y1084, Y1088, and Y1090
(13), R. Young via L.McReynolds; GM2163 (14), M. Marinus; MC1061
(15), R.Neve.HR110-HR112 were from H. Revel and are K-12/F+(X);
HR111 is also rglA-, and HR112 is rglA- rglB- (5, 6); HR111and
HR112 are also PI' and sup' (H. Revel, personalcommunication). K802
is a conjugational descendant of C600and Hfr Cavalli (9) and
probably carries the hsd region of HfrCavalli and the purB region
of C600. It is phenotypicallyRglA- RglB- (and thus presumably rglA
rglB) and McrA-McrB-. LCK8 (lac3 or lacYl galK2 galT22 metBI
hsdR2zj202::TnlO supE44; from L. Comai via B. Bachmann) isK802 with
a TnlO insertion between hsd and serB and has therestriction
phenotype of K802. By definition established inthis work, LCK8 and
K802 carry the mcrBl allele. ER1370(trp3l his] argG6 rpsLl04 tonA2
A(lacZ)rl supE44 xy17 mtl2metB1 serB28) is descended from JC1552
(7) via NK7254 (16)and several additional transductional steps.
Utilizing theclose proximity of mcrB to hsd, zy202::TnJ0, and
serB,mcrBl derivatives of ER1370, W3110, MM294, JM107, andY1084
were constructed, yielding some strains used in thiswork: ER1378
(mcrBl), ER1380 (mcrB1 zj202::TnlO) andER1381 (mcrB+) (all hsdR2
serB+, from ER1370); ER1398(from MM294, hsdR2 mcrBl); and ER1451
(from JM107,hsdR2 or R17, mcrBl). These constructions will be
detailedelsewhere (E.A.R., R. Trimarchi, and H. Revel,
unpublishedresults). Xr and Pl1i, were from our collection.
Transduc-tions were by standard methods (17).
Methylase Titrations. Five units (as defined by the
manu-facturer) of methylase (Alu I, Msp I, Hha I, Hph I, Taq I,dam,
or Hpa II; obtained from New England Biolabs) wasadded to 0.9 ,ug
of pBR322 DNA in a total vol of 30 ,ul; thissample was diluted
serially by factors of 2 into additionalDNA samples, such that the
total volume (15 ,ul) and amountof DNA (0.45 ,Ag) were maintained
in all samples. Forconvenience, the concentration of methylase
present in themost dilute sample was arbitrarily designated "1
unit" inFigs. 1 and 2; this is not the same as the manufacturer's
unit.Two control samples were carried through the procedureunder
the same conditions but lacked either methylase
orS-adenosylmethionine. Buffer conditions were 66 mM
Abbreviation: M. Hae II, modification methylase associated with
theHae II endonuclease; other methylases are designated
accordingly.
9070
The publication costs of this article were defrayed in part by
page chargepayment. This article must therefore be hereby marked
"advertisement"in accordance with 18 U.S.C. §1734 solely to
indicate this fact.
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Proc. Natl. Acad. Sci. USA 83 (1986) 9071
Tris HCl, pH 7.5/6.6 mM EDTA/0.33 mM 2-mercaptoetha-nol/0.15 mM
S-adenosylmethionine. Incubation was for 1 hrat 370C, and the
reaction was stopped by heating to 650C for10 min.Phage Growth and
Plating Tests. Phage stocks were pre-
pared by confluent lysis. Hae II-modified X was obtained
bygrowth on LCK8/pHaeII 4-11 (see below). Phage weretitered on
bacteria grown in XYM on X plates (18). E.O.P. isthe ratio of the
titer on the tested strain to the titer on LCK8.
Plasmids. Clones carrying the following modificationmethylases
were isolated by G.W. and coworkers (unpub-lished data): Alu I, Ava
I, Ava II, BamHI, Ban I, Ban II, BglI, Dde I, EcoRI, FnuDII, Hae
II, Hae III, Hga I, HgiAI, HhaI, Hha II, HindII, HindIII, Hinfl,
Hpa II, Msp I, Nla III, NlaIV, Pst I, Sal I, Taq I. All are carried
on pBR322 (19) orpUC19 (11). pHaeII 4-11 carries both the Hae II
endonucle-ase and methylase. pER82 was constructed from pHaeII
4-11by in vitro deletion of a portion of the endonuclease
gene.pPvuMl.9 carrying the Pvu II methylase was a kind gift fromR.
Blumenthal (12).
RESULTSRejection of a Clone Carrying the Hae H Methylase
Gene.
The plasmid pHaeII 4-11 carries the complete Hae II
restric-tion-modification system [recognition sequence, RGCGCY(R =
puRine and Y = pYrimidine); see ref. 20] cloned intopBR322; it was
isolated by using E. coli strain RR1 as host.The DNA from cells
containing pHaeII 4-11 is completelyHae II-modified. pHaeII 4-11
could be efficiently trans-formed into LCK8, an unrelated strain
derived from K802;however, neither pHaeII 4-11 nor pER82, a
derivativeencoding only the methylase, M. Hae II, could be
efficientlytransformed into several other laboratory strains ofE.
coli K-12, including ER1380, ER1381, JM107, MM294, and
W3110.Strains that rejected these plasmids could be transformed
bythem at a frequency of -10-5 relative to transformation of
apermissive strain. The rare transformants were of two types:those
carrying deleted plasmids lacking methylase activity,and those
carrying host mutations that eliminated the rejec-tion phenotype
permanently. We designated such host mu-tants mcrB- (for methyl
cytosine restriction; see below formcrA). In all subsequent work,
the response to pER82,carrying M. Hae II, or to Hae JI-methylated
Xvir (see below),was used to define the mcrB genotype. A detailed
geneticanalysis of this locus, adjacent to hsd, will be
publishedelsewhere (E.A.R., R. Trimarchi, and H. Revel,
unpublisheddata).McrB Phenotypically Restricts Hae II-Modified X.
The
plating efficiency of Hae II-methylated Xvir was comparedwith
that of unmethylated Xvir on isogenic mcrBl and mcrB+strains
(ER1378 and ER1381, respectively). Unmethylated Xplated with equal
efficiency on both strains (Table 1, column1). Hae JI-methylated X
phage plated at an efficiency of 0.03on the McrB+ strain when
compared with plating on theMcrB- strain (column 2). The
unmethylated progeny oftheseHae II-methylated phage (obtained by
passage in the absenceofM. Hae II) once again plated normally on
the McrB+ strain(column 3). This reacquisition of resistance to
restriction wasvery efficient: four of four independent progeny
stockspassaged in the absence of the Hae II methylase plated
Table 1. E. coli K-12 restricts Hae II-modified X
Plating efficiency relative to LCK8Of phage stock
On host strain XAC600 XALCK8/pHaeII 4-11 XALCK8
normally on the McrB+ strain. These progeny also
regainedsensitivity to Hae II restriction (not shown),
demonstratingloss ofHae II-specific methylation. Further passage
througha strain carrying M. Hae II restores sensitivity to
McrBrestriction at high efficiency (not shown). This
reversibleepigenetic alteration in plating properties is formally
identicalto classical host-controlled restriction (1-4).McrB
Restriction Is Methylcytosine Dependent. Samples of
pBR322 DNA were methylated in vitro to increasing extentswith
purified M. Alu I (which creates the sequence AGmCT),and were
transformed into ER1381 (McrB+) and its McrB-sibling, ER1378 (Fig.
1). The efficiency oftransformation intoER1381 was found to be
inversely related to the degree ofmethylation, while with ER1378
the efficiency of transfor-mation was independent of the degree of
methylation. Max-imal restriction by the McrB+ strain occurred when
the DNAwas fully protected from Alu I digestion (arrow).
Similarexperiments using other purified methylases (Table 2)
sug-gest that sensitivity to McrB restriction results from
cytosinemethylation but not from adenine methylation.A Second
Methylcytosine-Specific Restriction System Exists.
The McrA restriction system was recognized when wetransformed
mcrB mutants with pBR322 methylated in vitroby M. Hpa II (CmCGG).
We found restriction of thissubstrate to be independent of the mcrB
genotype of thestrain (Fig. 2). Both ER1378 (mcrBl) and ER1381
(mcrB+)(triangles) restricted Hpa II-methylated pBR322;
however,neither JM107 (mcrB+) nor its mcrBl derivative,
ER1451,restricted Hpa II-methylated pBR322 (circles).
Therefore,restriction of Hpa II-methylated DNA can be
eliminatedgenetically without eliminating McrB restriction and
viceversa. The locus encoding McrA is nearpurB (E.A.R. and
E.Latimer, unpublished data).
McrB-1.0
0C
0
0EI
0, McrB~0.01 1E ~~--'
12 4 8 16 32 64
[A/u I Methylase] (Arbitrary Units)
FIG. 1. McrB restriction of pBR322 DNA methylated in vitro atAlu
I sites. Samples (0.04 ,g) of pBR322 DNA, methylated toincreasing
extents with Alu I methylase, were transformed (8) inquadruplicate
into ER1381 (mcrB+ mcrA+) and ER1378 (mcrBlmcrA+). The average
number of transformants per plate from thecontrol with no added
methylase was 87 (ER1378) and 473 (ER1381).These values were taken
as transformation efficiencies of 1. Averagevalues for all other
samples were normalized to these values. Thetransformation
efficiency is plotted versus the concentration (inarbitrary units)
ofAlu I methylase. Error bars are standard deviationof the mean;
for clarity of presentation, the standard deviation is notshown for
ER1378. v, ER1381; v, ER1378. Portions of eachmethylated sample
were digested with Alu I endonuclease andelectrophoresed in a 1%
agarose gel to assess extent of methylation;complete protection
from digestion was achieved at the concentra-tion of methylase
indicated by the arrow.
ER1381 McrB+ 0.82 0.03 0.69ER1378 McrB- 0.88 1.2 0.91
Genetics: Raleigh and Wilson
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9072 Genetics: Raleigh and Wilson
Table 2. Methylated sequences restricted or not restricted by
mcrB
Methylatedsequence*Methylase
Hoststested Methylase
Methylatedsequence*
I. Restricted by McrB+ strainsA. Methylases of known
specificityAlu Itt AG CTDde It OTNAGHae UiPt GG CCHha Itt G CGCMsp
Itt CCGGPvu it CAG CTG
B. Methylases of unknown specificityAva It CYCGRGBan It GGYR
CCBan iHt GRG CYCHae Hit RG CGCY or RGCG 6YBglIt G CCN(5)GGC or
GCCN(5)GG CFnuDIIt CG 6GHgaIt G CGTC and/or GACG CHgi At G(A/T)G
6(A/T)CNla IIIt CATGNla IVt GGNN 6C
D, EDA, B, D, EA, BA, B, D, EE
DA, EA, DA, B, C, D, EA, DDA, DA, DDD
II. Not restricted by McrB+A. Methylases of known
specificitydamt G ATCEcoRIt GA ATTCHha lIt G ANTCHindIlt GTYR
ACHindIIIt A AGCTTPst It CTCG AGSal It GTCG ACTaq Itt TCG ABamHItt
GGAT CCdcm C C(A/T)GGHph It T CACCHpa IIt# C CGG
B. Methylases of unknown specificityAva Ilt GG(A/T)CCHinfIt
GANTC
pBR322 derivatives carrying cloned methylase genes (t) or pBR322
DNA methylated in vitro with purified methylase enzymes (t)
weretransformed into various strains of E. coli K-12. Clones
classified as not restricted transformed McrB+ and McrB- strains at
approximately thesame frequency; clones classified as restricted
transformed McrB+ hosts at a frequency of 10-3, or less, compared
to McrB- hosts. In vitromethylated pBR322 was classified as
restricted if the transformation efficiency dropped by at least a
factor of 10 as the DNA became fullymethylated. Recognition sites
for the methylase enzymes are assumed to be the same as the sites
recognized by the cognate endonuclease andare from ref. 21;
specific methylation sites are from ref. 22, except for dem (23),
Pvu II-(12), Dde I (J. Brooks, personal communication).
Therecognition sites are aligned at the position of known or
proposed methylation. The dcm methylase has not been tested as a
clone, but the hostis dcm' (as well as dam') and must, we assume,
find this methylation acceptable. Letters refer to sets ofmcrB1 and
mcrB- hosts. Set A: HR110(McrA+ McrB+), HR111 (A- B+), and HR112
(A- B-). Set B: C600 (A- B+) and K802 (A- B-). Set C: JM107 (A- B+)
and ER1451 (A- B-).Set D: MM294 (A' B+) and ER1398 (A' B-). Set E:
ER1381 (A' B+) and ER1378 (Al B-). Mcr phenotypes were defined on
the basis ofacceptance of the Hae II methylase plasmid for McrB and
of Hpa II-methylated pBR322 for McrA. HR110 is wild-type K-12;
HR111 (rglA)and HR112 (rglA- B-) are nitrosoguanidine-induced
derivatives of HR110. The McrB- strains in sets C-E are
transductants carrying the mcrBIallele found in LCK8; all of these
strains are also RglB-. The sets include at least two mcrA-
alleles: that introduced by nitrosoguanidinemutagenesis into HR111,
and that (those) found in C600 and JM107. C600 and JM107 are also
phenotypically RglA-; RglB restriction is observed,although it is
not as strong as that seen in HR111.*Inferred from the recognition
sequence of the endonuclease and the known (^) or proposed (')
position of methylation within that sequence.tTested for ability of
cloned methylase gene to transform.*Tested for ability of in vitro
methylated pBR322 to transform.
Specificity of McrB. The sequence specificity of the
McrBrestriction function was examined further using transforma-tion
by plasmids carrying cloned modification methylasegenes (Table 2).
Many of the methylases that confer sensi-tivity to McrB restriction
(part IA) are known to generate thesequence GmC. For the rest of
the mnethylases that confersensitivity, the site of methylation is
unknown (part IB); inthese cases, the sequence GmC potentially
could be gener-ated. The methylases that do not confer sensitivity
(part II)are either adenine methylases or are known not to
generatethe sequence GmC. The simplest conclusion is that GmC orRmC
is a necessary component of the McrB recognition site.Mcr and Rgl
Are Probably Identical. At least two indepen-
dent rglA mutations and two independent rglB mutationswere
present among the strains tested in Table 2. All rglBmutants
displayed the McrB- phenotype and all rglA mutantsdisplayed the
McrA- phenotype. This coincidence leads us tosuspect that Rgl and
Mcr are identical.The specificity of Rg1B from E. coli B differs
from that of
K-12, and its effect is substantially weaker (5, 6). We
expectthat the McrB function will, similarly, have a
differentspecificity and a weaker activity. With the exception of
M.Hpa II, the methylase gene clones referred to above wereisolated
by using RR1 as host; this hybrid strain carries therglB region
from E. coli B and is phenotypically McrB-,consistent with our
expectation. The M. Hpa II gene couldnot be cloned in RR1, but it
could be cloned K802. This isconsistent with the RglA+/McrA+
phenotype of RR1, andthe RglA-/McrA- phenotype of K802.
Survey of Common Laboratory Strains of E. coli K-12.Table 3
summarizes the results oftests of the Mcr phenotypesof various
strains commonly used in E. coli genetics and inmolecular cloning
experiments. All tested strains commonlyused for cloning
experiments carry at least one ofthe two Mcrsystems. The results
described above suggest that restrictionproblems may be encountered
when these strains are usedand the DNA to be cloned contains
methylcytosine.
DISCUSSIONWe show here that E. coli strains biologically
restrict DNAmethylated at some cytosine residues. Plasmids
carryingcloned methylases (Table 2), phage methylated in vivo
(Table1), or plasmids methylated in vitro (Figs. 1 and 2; Table 2)
areall restricted. We regard the phenomenon as restrictionbecause
it fits the classical definition: rejection, at least ofHae
II-modified X, depends upon the host that the phage aregrown on and
is reversible at very high frequency by growthof the phage on
another host (Table 1; see text).The observed restriction requires
methylcytosine in spe-
cific sequences. That pBR322 DNA methylated in vitro
isrestricted demonstrates that restriction is not dependent
onunknown products of, or sequences present in, cloned DNAfrom
foreign species; nor are alterations ofDNA other thanmethylation
required. We assume that the methylation re-quired is at the 5
position of cytosine. Of the methylases thatconfer sensitivity,
only M. Msp I has been shown to meth-ylate the 5 position (24, 25).
It has recently been shown that
Hoststested
CA, B, DA, B, DA, DDA, B, DDA, C, D, EA, B, DAll dcm'EA, C, D,
E
DA, D
Proc. Natl. Acad. Sci. USA 83 (1986)
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Proc. Natl. Acad. Sci. USA 83 (1986) 9073
c
.)4-
o 0.1L-E O
o0
enc
0.01
0
0
0 McrA0
v
V
0
V
124 8 16 32 7 64
[Hpo II Methylase] (Arbitrary Units)
FIG. 2. McrA restriction of Hpa I-methylated pBR322 DNA.Hpa
JI-methylated pBR322 was transformed in duplicate intoER1381 (mcrB+
mcrA+), ER1378 (mcrB- mcrA+), JM107 (mcrB+mcrA-), and ER1451
(=JM107 mcrBI), and the transformationefficiencies at various
methylation levels were determined as de-scribed in the legend to
Fig. 1. v, ER1381; v, ER1378; e, JM107; o,ER1451. Arrow indicates
complete protection from Hpa II endonu-clease digestion, as in Fig.
1.
some methylases modify the N4 position of cytosine (25, 26),and
we cannot rule out the possibility that such methylationalso
confers sensitivity.We have identified two different restriction
specificities,
McrA and McrB. These specificities are genetically
different,since mutations that eliminate one activity have no
effect onthe other activity. Cytosine methylases exist that do
notconfer sensitivity to either Mcr system (Ava II, BamHI, dcm,Hph
I); the systems must therefore discriminate amongmethylated
residues. We suggest that the McrB functionrecognizes the consensus
sequence of GmC, or RmC. Therecognition sequence of the McrA
function remains uncer-
Table 3. Mcr phenotypes of selected laboratory strains
Mcr phenotypeStrain A B
K-12 + +W3110* + +MM294* + +ER1370* + +JM101 + NTJM107* - +C600
- +Y1084* - +Y1088 -t +Y1090 -t +LE392 - +CR63 - +HB101 + -RR1 +
-MC1061 NT -K802 - -GM2163 - -
NT, not tested.*mcrB derivatives are available.tInferred from
phenotype of plasmidless ancestor.
tain, since only one methylated sequence, CmCGG, has beenfound
to confer sensitivity. Since RR1, the host used forcloning most of
the methylase genes, is McrA' McrB-, ourcollection probably
contains only methylases that are insen-sitive to McrA activity.The
identity of Mcr and Rgl functions is suggested by the
facts that both systems are active only on DNA that
containsmodified cytosine, and that among the strains tested, Mcr
andRgl phenotypes invariably coincided. Genetic analysis(E.A.R., R.
Trimarchi, and H. Revel, unpublished data)shows that both rglB and
mcrB mutants map close to hsd, at99 min; that mutants isolated for
either phenotype exhibit theother; and that the two phenotypes are
not separated bytransduction. In addition, both McrA and RglA are
affectedby a mutation that maps near purB (E. Latimer and
E.A.R.,unpublished data).The Mcr restriction systems present a
potentially serious
problem for those engaged in molecular cloning cf foreignDNA
into E. coli. Possession ofboth systems is the wild-typestate,
since the original K-12 strain carries both, 4nd manycommon
laboratory strains of E. coli K-12 carry at least oneof the systems
(Table 3). The DNA of many organismscontains methylated cytosine
(27) and consequently shouldbe sensitive to Mcr restriction, at
least at some sites. WhenpBR322 is fully methylated with the Alu I
methylase, -2% ofits cytosine residues are methylated-a level of
methylationwell within the range found in eukaryotic organisms-and
thenumber of transformants in McrB+ strains is reduced to 1%of the
level found with unmethylated DNA. Our resultssuggest that the use
of strains defective in both Mcr functionswould enhance recovery of
transformants carrying somesequences from ligation mixtures
containing genomic DNA.We do not know the molecular mechanism of
M4r restric-
tion. The enzymology of Rgl restriction also remai s uncer-tain.
A function present in RglB+ cells, but not in RgB- cells,has been
shown to act on hemihydroxymethylated fd RFDNA: an Rg1B+ RecBC-
extract altered the mobility of thesubstrate in a sucrose gradient
and rendered it sensitive toRecBC exonuclease activity from an
Rg1B- extract (28, 29).However, a partially purified fraction that
rendered thesubstrate sensitive to RecBC did not alter its mobility
ongradients; a heat-labile non-dialyzable factor from an
RgIB-extract was required for this. It is possible that RglB
functionrequires two or more different subunits, or that a
secondindependent enzymatic activity is required for
substrateconversion to the RecBC-sensitive form. In many other
casesof phenotypic restriction, restriction is mediated by
site-specific double-strand cleavage of the target DNA.
Othermechanisms are possible, however. For exaniple, theuracil-DNA
glycosylase (ung) ofE. coli, in combination withapyrimidinic
endonucleases, can mediate restriction ofuracil-containing DNA
(30).Two other methyl-specific restriction systems have been
reported. The best characterized system, Dpn I, restrictsphage
in vivo, and consists of an otherwise ordinary restric-tion
endonuclease that specifically recognizes and cleaves amethylated
sequence (GmATC). In this case, the "modifi-cation" that protects
DNA from cleavage is the absence ofmethylation at that sequence
(31, 32). Several endonucleaseswith the same specificity have been
found in bacteria otherthan the original Diplococcus pneumoniae
species (21). Theother system, from Acholeplasma laidlawii, has so
far notshown evidence of sequence specificity. It results in
pheno-typic restriction of DNA that has been methylated in vivo
orin vitro with any of several sequence-specific cytosinemethylases
(33).The existence of methyl-requiring restriction systems en-
sures that methylation alone is not sufficient for
defenseagainst restriction. Many phage use active
antirestrictionmechanisms (34) or extensively modify their DNA,
some
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9074 Genetics: Raleigh and Wilson
with quite baroque substitutions (glucose, putrescine,
gluta-mine, isopentene; see ref. 35), but very few have
limitedthemselves to the relatively simple expedient of
incorporat-ing a methylated base in place of the normal one.
Thissuggests that systems specific for simple modifications maybe
common. Demonstration of two previously unrecognizedmethyl-specific
restriction systems in the well-studied orga-nism E. coli K-12
suggests that such restriction may indeedbe much more widespread
than previously appreciated. Thepaucity of restriction systems that
are known to requiremethylated DNA may reflect primarily the use of
unmethyl-ated substrates in the search for restriction
endonucleases,rather than the rarity of such systems.
The authors thank Helen Revel for strains; Barbara Bachmann,Jon
Beckwith, Ashok Bhagwat, Bob Blumenthal, Joan Brooks, DonComb,
Nancy Kleckner, Noreen Murray, Mike Nelson, HelenRevel, Denise
Roberts, Rich Roberts, and Ira Schildkraut fordiscussion and for
critical review ofthe early draft ofthis manuscript;Bob Blumenthal
and Antal Kiss for communication of results priorto publication;
and Russ Camp, Chuck Card, Rebecca Croft, TinaJager, Elizabeth
Latimer, Keith Lunnen, Chris Taron, and RuthTrimarchi for help with
transformation and strain construction.
1. Luria, S. E. & Human, M. L. (1952) J. Bacteriol. 64,
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