Proc. Natl. Acad. Sci. USAVol. 86, pp. 9223-9227, December 1989Biochemistry

DpnA, a methylase for single-strand DNA in the Dpn II restrictionsystem, and its biological function

(methyltransferase specificity/DNA sequence recognition/genetic transformation/plasmid transfer/Streptococcus pneumoniae)

SUSANA CERRITELLI, SYLVIA S. SPRINGHORN, AND SANFORD A. LACKSBiology Department, Brookhaven National Laboratory, Upton, NY 11973

Communicated by Hamilton 0. Smith, September 11, 1989 (received for review July 5, 1989)

ABSTRACT The two DNA-adenine methylases encoded bythe Dpn II restriction gene cassette were purified, and theiractivities were compared on various DNA substrates. DpnAwas able to methylate single-strand DNA and double-strandDNA, whereas DpnM methylated only double-strand DNA.Although both enzymes act at 5'-GATC-3' in DNA, DpnA canalso methylate sequences altered in the guanine position, but ata lower rate. A deletion mutation in the dpnA gene wasconstructed and transferred to the chromosome. Transmissionby way of the transformation pathway of methylated andunmethylated plasmids to dpnA mutant and wild-type recipi-ents was examined. The mutant cells restricted unmethylateddonor plasmid establishment much more strongly than didwild-type cells. In the wild type, the single strands of donorplasmid DNA that enter by the transformation pathway areapparently methylated by DpnA prior to conversion of theplasmid to a double-strand form, in which the plasmid wouldbe susceptible to the Dpn II endonuclease. The biologicalfunction of DpnA may, therefore, be the enhancement ofplasmid transfer to Dpn TI-containing strains of Streptococcuspneumoniae.

The Dpn I and Dpn II restriction systems are found indifferent strains of Streptococcus pneumoniae. They areencoded by alternative genetic cassettes located at the sameposition in the chromosome (1). Dpn I is an unusual restric-tion endonuclease in that it only acts on methylated sites inDNA (2). The two systems are complementary and mutuallyexclusive since DNA from a Dpn I strain, which is notmethylated at 5'-GATC-3' sites, is susceptible to Dpn II, andDNA from a Dpn II strain, which contains 5'-GmATC-3', issusceptible to Dpn I (3). Thus, when grown on the comple-mentary strain, bacterial viruses are reduced in infectivity toa level <10-5 (4). Existence of the complementary systemsmay enhance survival of the species with respect to viralepidemics (5). The susceptibility of viruses to restrictionresults from their introduction into the cell of their double-strand DNA. Dpn I and Dpn II act only on double-strandDNA, when both strands are methylated and unmethylated,respectively, at 5'-GATC-3' sites; neither enzyme acts onsingle-strand DNA or on hemimethylated double-strandDNA, in which one strand is methylated and the other strandis not (6).Chromosomal transformation is not affected by the Dpn

restriction enzymes in the recipient cell. Cells are trans-formed with respect to a chromosomal marker at the samefrequency, whether or not the donor DNA is methylated, inboth Dpn I- and Dpn II-containing recipients (1, 7). This lackof restriction effect presumably reflects the molecular fate ofDNA in transformation: neither the single strands that enterthe cell (8) nor the hemimethylated heteroduplex DNA that

results from integration into the chromosome (9) is suscep-tible to the restriction enzymes.

Plasmid transfer in S. pneumoniae by way of the transfor-mation pathway for DNA uptake is only mildly restricted bythe Dpn I and Dpn II systems, with transfer reduced to -40%in the cross-transformation (7). Greater susceptibility ofplasmid transfer than of chromosomal transformation torestriction would be expected from the need to reconstitutedouble-strand plasmids from the single-strand fragments thatenter the cell (10). Plasmid establishment presumably re-quires considerable new synthesis to repair gaps in thereconstituted structure (10). In the case of transfer of anunmethylated plasmid to a Dpn II-containing recipient, suchsynthesis would create susceptible sites. Therefore, it ispuzzling that the restriction effect observed on plasmidtransfer is so slight.The Dpn II cassette contains three genes, dpnM, dpnA,

and dpnB (1), that encode, respectively, two DNA methyl-ases, DpnM and DpnA, and the Dpn II endonuclease (11). Allthree enzymes recognize 5'-GATC-3' sequences in DNA, butDpnA appeared to be less constrained than the others in itsspecificity (3, 11). In this work, an improved procedure forpurifying large amounts of the DpnA methylase was devised.Its substrate specificity was examined and compared to thatof the other methylase, DpnM. Furthermore, a deletionmutation of dpnA was constructed and introduced into thechromosome. The effects of the mutation on restriction ofplasmid transfer and chromosomal transformation were de-termined.

MATERIALS AND METHODSBacterial Strains and Plasmids. Strains and plasmids of S.

pneumoniae (1, 7, 12) and Escherichia coli (11) were previ-ously described except for those constructed in this work.Growth and Transformation of Bacteria. Cultures of S.

pneumoniae were grown in semisynthetic medium (13); theywere treated with DNA and selected for chromosomal trans-formation or plasmid establishment as described (13, 14).Novobiocin-resistant (Nov9 transformants were selectedwith novobiocin at 10 ,g/ml and chloramphenicol-resistant(Cm9 transformants were selected with chloramphenicol at2.5 ,ug/ml. To screen for Cm-sensitive (Cms) transformants,we adapted a method used for maltose-negative (Mal-) clones(13); with 0.2% sucrose and chloramphenicol at 1.0 ,ug/ml,Cms clones gave small colonies whereas Cmr clones gavelarge colonies.

Procedures used for the growth and transformation of E.coli and for hyperexpression ofDpn II system enzymes havebeen reported (11). The latter depended on induction of T7RNA polymerase in BL21(DE3) hosts (15).

Abbreviations: Nov, novobiocin; Cm, chloramphenicol; Str, strep-tomycin; Mal, maltose; Tc, tetracycline; r, resistan(t)(ce); s, sensi-tive; ds 12-mer, 5'-CGCGGATCCGCG-3'; ss 12-mer, 5'-ATTA-GATCGCCG-3'.

9223

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9224 Biochemistry: Cerritelli et al.

Enzyme Purification and Assay. The method used forpurifying DpnM has been described (16). Details of themodified procedure for purifying DpnA were similar, withexceptions as noted. Methylase assays, in which [14C]methylgroups were transferred from S-adenosylmethionine toDNA, were carried out as described (17).

Preparation and Manipulation of DNA. Methods used forpreparation of plasmid and chromosomal DNA were previ-ously indicated (11). Thymus DNA was obtained commer-cially. M13mp8 DNA was prepared (18) from phage grown onE. coli K440, a dam-3, F+ strain, kindly provided by M.Marinus (University of Massachusetts, Worcester). Oligo-nucleotides were synthesized by the phosphoramiditemethod on a Systec Microsyn 1450 DNA synthesizer. DNAmanipulations in vitro were carried out by standard methods(19). For a review of in vivo recombination mechanisms in S.pneumoniae transformation and plasmid transfer, see ref. 20.

Construction of dpnA Mutant Strains. A deletion mutationof dpnA was constructed in vitro using pLS211 (Fig. 1). Theplasmid was partially digested with Dra I to remove a389-base-pair (bp) fragment entirely within the coding regionfor DpnA (1). An Nhe I linker (5'-GGCTAGCC-3') wasligated into the site of the deletion to give pLS259. Themutated dpnA segment was transferred to a streptococcalplasmid by ligating the 1.9-kilobase (kb) Nco I-BamHI frag-ment of pLS259 with the vector-containing 6.9-kb Nco I-BamHI fragment of pLS202 (1) to give pLS275. The mutantallele was called dpnA275.To assist the transfer of dpnA275 to the chromosome, we

used a cat gene marker conferring Cmr (12), which had beenpreviously inserted into an EcoRV site 96 bp upstream of theDpn I cassette to give pLS281 (21). On transfer of pLS281 toa Dpn II strain, 764, chromosomal facilitation of plasmidestablishment (14) substituted the Dpn II cassette down-stream from the cat marker, giving rise to pLS283. Bytransformation the cat marker was introduced from pLS281and pLS283, respectively, into the chromosomes of the DpnI strain 1133 and the Dpn II strain 1134 (illustrated in Fig. 1).The presence of the cat insert had no effect on expression ofDpn I (21) or Dpn II system genes (this work; data notshown). These derivatives enabled us to obtain plasmidclones of Dpn I and Dpn II with greater lengths of chromo-somal DNA on either side of the restriction gene cassette. Toaccomplish this, we cloned the 7.0-kb BamHI fragment fromthe chromosome of strain 1133 into pLS10 (22) to givepLS402 (Fig. 1). Introduction of this plasmid into strain 764gave pLS411 by chromosomal facilitation. The 3.9-kb Pst Ifragment of pLS411 was used to transform plasmid pLS275to give pLS395, which rendered the cells tetracycline resist-ant (Tcr). In pLS395 the tet gene of the vector is restored,

chromosomal homology to the left of the Dpn II cassette isincreased, yet the dpnA275 mutation is retained (Fig. 1).The dpnA275 mutation was introduced into the chromo-

some of strain 1134 by transforming with pLS395 and select-ing for the Cms strain 1135. The proximity of the dpnAmutation to the cat insertion site resulted in the presence ofthe mutation in most of the Cms transformants. To confirmthe presence of dpnA275 in the chromosome, strain 1135 wastransformed to Tcr using Sac I-cleaved pLS402, which forcedchromosomal facilitation and transfer of the Dpn II cassetteto the plasmid; the resulting plasmids were examined for thepresence of the additional Nhe I site. A different route wasused to introduce dpnA275 into the chromosome to makestrain 1137. First, the cat marker was transferred into pLS395to give pLS397. The latter then transformed 733 to Cmrtogether with dpnA275. Finally, the cat gene was removed bytransformation with pLS395 to give 1137. Although dpnA275blocked DpnA activity in methylation of single-strand DNA,the mutation had no effect on the levels of DpnM methylaseor Dpn II endonuclease expressed by cells or plasmidscarrying the mutant allele (data not shown).

RESULTSPurification of the DpnA Methylase. An earlier purification

procedure (11) was modified to prepare larger amounts ofDpnA. After hyperexpression in the BL21(DE3) (pLS211)system, variable and often large amounts of the Dpn proteinsin extracts were insoluble (Fig. 2a). DpnA precipitates at lowsalt concentration, and for this protein the insoluble fractioncould contain as much as 80% of the total amount present.DpnA was purified, therefore, from both soluble and insol-uble fractions. A 2-liter culture yielded -1 mg of electro-phoretically pure DpnA protein.The soluble extract was treated with bovine DNase I and

passed over a column of DEAE-Sepharose CL-6B (Pharma-cia) as described for DpnM (16). The breakthrough materialwas then fractionated on heparin-agarose (Bio-Rad) to sep-arate DpnA from DpnM (Fig. 2b). Further purification byagarose gel filtration on Superose 12 (Pharmacia) gave DpnAfree of other detectable proteins (Fig. 2c).The insoluble fraction, which sedimented during centrifu-

gation at 17,000 x g for 30 min, was suspended in extractionbuffer containing 1 M NaCl. Most of the DpnA, but not otherproteins, dissolved, thereby resulting in partial purification ofDpnA (Fig. 2d). After dilution to 0.3 M NaCl and chroma-tography on heparin-agarose, considerable purity wasachieved, with some fractions devoid of contaminatingDpnM (Fig. 2e).

pLS211T P B

O ..-amp ori PT7

pET -5

pLS259T P B

rolomp ori PT7

C P DDB PT

dponM A B

C P NB PT

-i -BdponM B

pLS275T N T P P C P NB PT

moiM rep dpnM B

Strain Cat1 134 aBj TcX D DB PT T

dpnM A B

I1kb

pLS402 Cal

i I----p- II -I-0-rep let dpnC D

PLSIO T

T B

pLS41 v

B P P B TNC P DDB PT T B1~ ~~~ .

0__ _ 0

rep let dpnM A B

YpLS395 V

T N T P P B TN C P NB PT

-

_

mo/M rep tet dpnlM B

StrainB ,, B TN C P NB PTB- R - *-"M -1 10 1 1

dpnM B

BT

FIG. 1. Construction of a dpnA mutation and itstransfer to the chromosome. Plasmids are shown lin-earized with vector as thin line and dpn chromosomalsegment as heavy line. Chromosomal segments ofstrains are bordered by heavy dashed line. Intact genesare indicated with arrows showing direction of tran-scription. The cat gene insert was a Dpn I fragmentfrom pJS3. Paths of construction are shown by blockarrows: open, in vitro construction; filled, in vivoconstruction by transformation. Only some restrictionsites are shown, according to the following code: B,BamHI; C, Nco I; D, Dra I; N, Nhe I; P, Pst I; S, SacI; T, BstEII.

Proc. Natl. Acad. Sci. USA 86 (1989)

Proc. Natl. Acad. Sci. USA 86 (1989) 9225

(a) (b)123 12345

68 _

4329

14_

...

(c) (d) (e)2345

_" _

FIG. 2. Purification of the DpnA methylase. Samples of proteinat various stages of purification were subjected to SDS/poly-acrylamide gradient gel electrophoresis (23) and stained withCoomassie blue. Amounts of fractions applied are expressed in termsofequivalent volumes of the induced culture of BL21(DE3) (pLS211),which contained 0.3 mg of protein per ml. (a) Crude extract. Lane 1,protein markers (indicated by scale in other gels), 1 gg each; lane 2,pellet, 0.1 ml; lane 3, supernatant, 0.1 ml. (b and c) Successive stepsin purification of the supernatant enzyme. (b) Heparin-agarose frac-tions. Lanes 1-5, even fractions 34-42, 2 ml. (c) Superose gel filtrationfraction 53, 4 ml. (d) Pellet dissolved in 1 M NaCl, 1 ml. (e)Heparin-agarose fractions of dissolved pellet. Lanes 1-5, even frac-tions 22-30, 2 ml. Arrows indicate positions expected for DpnA(upper) and DpnM (lower).

Action of DpnA on Single-Strand DNA. The methylatingactivity of purified DpnM and DpnA was compared with avariety ofDNA substrates unmethylated at 5'-GATC-3' sites.These included double-strand DNA from calfthymus, single-strand DNA from phage M13mp8, and synthetic oligodeoxy-nucleotides 5'-CGCGGATCCGCG-3' (ds 12-mer) and 5'-ATTAGATCGCCG-3' (ss 12-mer). The first oligonucleotideis self-complementary, and =90% was in a double-strandform after gel electrophoresis; the second appeared to be onlyin a single-strand form (results not shown).Both methylases are active with thymus DNA, although

DpnM shows a somewhat higher specific activity (Table 1).Most strikingly, however, DpnA is highly active in methyl-ating the single-strand M13 DNA, even more than withthymus DNA. In contrast, DpnM showed no detectableactivity on M13mp8 DNA. Consistent with the specificity ofDpnA for single-strand and double-strand DNA is its highactivity on both oligonucleotides. DpnM showed high activ-ity with the double-strand oligonucleotide. With the "single-strand" oligonucleotide, however, DpnM showed about 5%of its activity on the double-strand oligonucleotide. Inasmuchas the four nucleotides constituting the recognition site arecomplementary, they may provide sufficient double-strandedness to the ss 12-mer to allow some action by DpnM.Conversely, the apparently higher specific activity of DpnAon the ds 12-mer than on thymus DNA may result from a

significant amount (10%) of the former remaining in a single-strand form.

Specificity of DpnA Methylation in Vitro. Earlier studiesshowed that DpnA methylated 5'-GATC-3' sites in vivo, butin vitro the enzyme methylated other sites, as shown by itsability to methylate DNA already methylated at 5'-GATC-3'sites (11). We therefore examined activity of the purifiedenzyme on a variety of oligonucleotide substrates containing5'-GATC-3' and related sequences. Table 2 presents resultswith a set of single-strand oligonucleotides varying in a single

Table 1. Comparison of DpnA and DpnM methylation ofsingle-strand and double-strand DNA

Methyl groupstransferred to

DNA, pmol/hr perDNA,t ,.g of enzyme*

DNA substrate Ag DpnM DpnA

Thymus DNA 16 320 97M13mp8 DNA 16 <3 870ds 12-mer 1 310 260ss 12-mer 1 15 430

*Values are averages of three or more determinations using 0.03-0.15 ,ug of enzyme. Amounts were chosen to give <50%16 ofmaximum reaction. Thus, the results, which were extrapolated to 1,.g of enzyme protein, represent rates of reaction.tThe approximate numbers of pmol of 5'-GATC-3' sites are 200, 50,and 250 for the thymus, M13mp8, and 12-mer substrates, respec-tively.

base of the recognition sequence. It is evident that DpnA canmethylate sequences other than 5'-GATC-3', when the al-tered base is in the position of guanine. However, themethylating activity on these deviant substrates is only 12%or less than with the canonical site. Alteration of bases in thethymine or cytosine positions reduces activity more com-pletely, although a little activity was observed on substitutionby the other pyrimidine.

In vivo DpnA methylates DNA at the N6 position ofadenine in 5'-GATC-3', as shown by susceptibility of themethylated DNA to Dpn I. The methylation of relatedsequences in vitro also appears to be at this position. Nomethylation was observed with oligonucleotides substitutedin the adenine position by either guanine, thymine, or cy-tosine (data not shown). Substitution of cytosine, the onlyother potentially methylatable base in the recognition se-quence, by thymine did not completely eliminate methylation(Table 2).

Effect of dpnA on Restriction of Plasmid Establishment.Isogenic pairs of strains carrying the wild-type and dpnA275mutant Dpn II gene cassettes in their chromosomes werecompared with respect to restriction of plasmid establish-ment and chromosomal transformation. The plasmids used-pJS3, pMV158, and pLS70-all contain the pLS1 replicon(24) and, respectively, 2, 8, and 11 5'-GATC-3' sites (12, 25).These plasmids, as well as chromosomal DNA, were pre-pared in unmethylated and methylated form from Dpn I- andDpn II-containing hosts, respectively. Transformation wascarried out with -0.1 ,ug of plasmid DNA (except forunmethylated pLS70, for which about half this concentrationwas used) or 1.0 ,ug of chromosomal DNA per ml of culture.

Table 2. Nucleotide sequence specificity for methylationby DpnAOligodeoxynucleotide pmol methylated Relative

substrate* per hr/,ug of DpnA action5'-ATTAGATCGCCG-3' 810 (1.00)5'-ATIAAAMGCCG-3' 100 0.125'-ATrACAICGCCG-3' 46 0.065'-ATTATACGCCG-3' 29 0.045'-ATTAQACCGCCG-3' 15 0.025'-ATTAQAACGCCG-3' <5 <0.015'-ATTAUAGCGCCG-3' <5 <0.015'-ATrAQAUIGCCG-3' 6 0.015'-ATTAjATGGCCG-3' <5 <0.015'-ATfAQAIAGCCG-3' <5 <0.01

Recognition sequence is underlined; altered bases are shown inboldface.*Five micrograms (1250 pmol) in assay mixture.

Biochemistry: Cerritelli et al.

VIW -.0-

-WWWY -.4-

L-

9226 Biochemistry: Cerritelli et al.

Comparison with results for transformation of the null re-striction strain, 762, affords a correction for variation inquality or quantity of the donor DNA. Selection for Cmr orTcr measured plasmid establishment; selection for Mal',streptomycin resistance (Str9), or Novr measured chromo-somal transformation.One pair of recipient strains was isogenic with strain 762.

These are 764, in which the Dpn II cassette replaced thedefective Dpn I cassette of762 (1, 4), and its derivative, strain1135. The other pair, 733 and 1137, are derivatives of theoriginal S. pneumoniae strain from which the Dpn II cassettewas obtained. All four recipients contain malM point muta-tions that allow measurement of chromosomal transforma-tion by the malMgene cloned in pLS70. The data obtained forstrain 762 and both isogenic pairs (with wild-type or mutantdpnA) are shown in Table 3. To calculate the restrictioneffect, the ratio of transformants with unmethylated relativeto methylated donor DNA was divided by the ratio given bythe null strain. These results are presented in Table 4. Theresults with the wild-type recipients, 764 and 733, confirmearlier work (7) by showing no restriction of chromosomaltransformation and only limited restriction, -50%, of plas-mid establishment.

Table 3. Effect of dpnA mutation on restriction ofplasmid transfer

Transformants x

10-3/mlt

Recipient Donorstrain* DNA

762 pJS3(null) pMV158

pLS70pLS70Chrom.Chrom.

764 pJS3(wild) pMV158

pLS70pLS70Chrom.Chrom.

1135 pJS3(A275) pMV158

pLS70pLS70Chrom.Chrom.

733 pJS3(wild) pMV158

pLS70pLS70Chrom.Chrom.

1137 pJS3(A275) pMV158

pLS70pLS70Chrom.Chrom.

Marker

Cmr

TcrTcrMal'StrrNovrCmrTcrTcrMal'Strr

NovrCmrTcrTcrMal'Strr

NovrCmrTcrTcrMal'StrrNovrCmrTcrTcrMal'Strr

Novr

Dpn Istraint

1.32.86.1

39090130

0.731.17.9

35042570.330.200.30

83084110

0.574.95.9

5,600250260

2.40.290.23

20,000320480

Dpn II

strain§0.802.617

1,200100140

0.751.6

341,100

50823.05.4

721,200530640

0.7410

1609,1002602102835

38049,000

760960

Dpn Idonor/Dpn II

donor

1.621.080.360.330.900.930.970.690.230.320.840.700.110.0370.00420.690.160.170.770.490.370.620.961.240.0860.00830.000610.410.420.50

Chrom., chromosomal DNA.*Dpn II genotype is shown in parentheses; strain pairs 764 and 1135and 733 and 1137 are isogenic.tNumber of transformants per ml with donor DNA from Dpn I orDpn II strain.tPlasmid host was 217; chromosomal DNA from 533; DNA wasunmethylated.§Plasmid host was 697; chromosomal DNA from 1138, nov trans-formant of 697; DNA was methylated.

Results for the dpnA275 recipient strains are markedlydifferent (Table 4). Establishment of unmethylated plasmidsis restricted to a level between 0.2% and 7% of methylatedplasmid establishment. The extent of restriction correlateswith the number ofDpn II restriction sites in the plasmids: 2for pJS3, 8 for pMV158, and 11 for pLS70. Plasmids withouthomology to the chromosome, such as pJS3 and pMV158, areestablished only by complementary plasmid strand reassem-bly (10, 14). However, inasmuch as pLS70 contains a largesegment ofDNA homologous to the recipient chromosome,most of its establishment must occur by chromosomal facil-itation rather than by direct plasmid reconstitution (14).Thus, restriction by Dpn II in dpnA mutants, which stronglyreduces transfer ofboth pMV158 and pLS70, must affect bothprocesses of plasmid establishment.A most plausible explanation for the severe restriction of

plasmid establishment in dpnA mutants, but not in wild-typerecipients, is that the methylation of incoming single-strandDNA by the DpnA methylase protects the plasmid sites fromlater attack by the Dpn II endonuclease. In the absence ofsuch methylation, attack by Dpn II would follow conversionofthe plasmid DNA to a double-strand unmethylated form by(i) association with a complementary donor plasmid strand,(ii) repair synthesis ofunpaired segments after partial plasmidreconstitution, or (iii) complementary strand synthesis aftercircular synapsis (20) of a plasmid strand with chromosomalDNA. The primary biological function of the DpnA methyl-ase may be to allow the transfer of plasmids from Dpn Istrains of S. pneumoniae or from other bacterial species toDpn II strains.Some restriction of chromosomal transformation by un-

methylated DNA may also occur in dpnA275 recipients(Table 3). This was not evident for the Mal' marker carriedby plasmid pLS70, but the two chromosomal resistancemarkers showed restriction effects to the level of 20-50%.Unless Dpn II can act to some extent on single-strand DNA,which has not been observed (6), an explanation for thiseffect is not apparent. However, to the extent that suchrestriction occurs, the DpnA methylase would enhance, also,chromosomal transformation.

DISCUSSIONThe DpnA methylase is unusual in its ability to methylatesingle-strand DNA. It appears to have evolved for thisparticular function inasmuch as the Dpn II restriction system,of which it is a part, already contains a potent methylase fordouble-strand DNA, DpnM. Its primary biological role maybe to enable plasmid transmission to Dpn II-containing cellsby way of the transformation pathway ofDNA entry, whichintroduces DNA in single-strand form (8). Methylation of theincoming strand would protect the subsequently reconsti-tuted plasmid from Dpn II cleavage. The mechanism ofchromosomal transformation itself assures its resistance torestriction (6). Thus, the systems of S. pneumoniae that allowthe species to benefit from genetic exchange and plasmidtransfer are largely immune to restriction, which is presum-ably directed at the prevention of bacterial virus infection.

Earlier work showed that methylated plasmids were littlerestricted in Dpn I-containing cells. This can be explained bythe resistance to Dpn I cleavage of reconstituted plasmidscontaining large regions of newly synthesized strands, whichwould be unmethylated (10). A similar explanation wasuntenable for the mild restriction ofunmethylated plasmids inDpn II-containing cells (7). As indicated above, however, thismild restriction can now be attributed to methylation byDpnA of the single-strand donor DNA that enters the cell byway of the transformation pathway.

Previously observed differences between restriction ofplasmid transfer in the conjugative and transformation path-

Proc. Natl. Acad. Sci. USA 86 (1989)

Proc. Natl. Acad. Sci. USA 86 (1989) 9227

Table 4. Summary of restriction effects on plasmid transferRestriction effect with isogenic recipient strain pairs

Transformed null strain Original Dpn II strain

Donor 764 1135 A275/ 733 1137 A275/DNA Marker (wild) (A275) wild (wild) (A275) wild

pJS3 Cmr 0.60 0.068 0.11 0.48 0.053 0.11pMV158 Tcr 0.64 0.034 0.053 0.45 0.008 0.017pLS70 Tcr 0.64 0.012 0.019 1.03 0.002 0.002pLS70 Mal+ 0.97 2.1 2.2 1.88 1.24 0.66Chrom. Stif 0.75 0.18 0.24 1.07 0.47 0.44Chrom. Novr 0.67 0.18 0.27 1.33 0.54 0.41

Restriction effect (first two columns for each pair of strains) was calculated as the ratio of unmethylated to methylateddonor DNA transformants (Table 3) divided by the null strain 762 ratio (Table 3). Chrom., chromosomal DNA.

ways can be explained by the action ofDpnA only in the lattercase. On transfer from a Dpn II strain to a Dpn I strain, theconjugative plasmid pIP501 was not restricted (W. Guild,personal communication), which is consistent with conjuga-tive transfer of only a single strand. As in the transformationpathway, replication ofthe complementary strand would givea hemimethylated duplex, which is not a substrate for Dpn I(6). In contrast, unmethylated pIP501 was restricted to 1i-4when transferred to a wild-type Dpn II strain (26). Thesmaller plasmid pMV158, when mobilized by pIP501, wasrestricted to 2%. In the case of conjugative single-strandtransfer, very rapid synthesis ofthe complementary strand byenzymes introduced with theDNA during conjugation, whichcan occur during conjugative transfer (27), presumably ren-ders the DNA susceptible to the Dpn II endonuclease priorto action of DpnA. In transformation, the less rapid recon-stitution and repair synthesis dependent on host enzymeswould allow time for DpnA action.

In addition to acting on single-strand DNA, DpnA differsfrom DpnM in its less stringent requirement for the 5'-GATC-3' target sequence. Recognition of adenine, thymine,and cytosine was critical for action, but variation of the firstbase, guanine, was tolerated to some extent. The signifi-cance, if any, of this degeneracy is unknown.Also unexplained at present is the apparent redundancy of

DpnM, inasmuch as DpnA itself can methylate 5'-GATC-3'in double-strand DNA. When the Dpn II system cassette ispresent on a multicopy plasmid containing intact dpnA anddpnB, the dpnM gene is not essential (11). It appears that amultiple dose of the dpnA gene expresses sufficient DpnAmethylase to protect cellular DNA from the Dpn II endonu-clease. Further investigation is necessary to determinewhether a single copy of dpnA in the chromosome will alsoallow the cell to dispense with DpnM.The amino acid sequence of DpnA is distinct from DpnM

(1); only three small boxes of similarity, common to a varietyof DNA adenine methylases (11, 28), are evident. Thesequence of the Hinfl methylase of Haemophilus influenzae,which methylates adenine in 5'-GANTC-3', was recentlyreported (29). DpnA is very similar to this protein; 40% of itsresidues are identical to the 255 amino-terminal residues ofthe Hinfl methylase. The two proteins are presumably ho-mologous in a biological sense-that is, descended from acommon ancestral form. However, DpnA does not methylate5'-GANTC-3' sequences in oligonucleotides, with the excep-tion of5'-GAATC-3' (data not shown). DpnM was previouslyfound to be homologous to the Dam methylase ofE. coli (30).Despite their adjacent location in the Dpn II gene cassette, itappears that dpnA and dpnM did not arise by gene duplicationbut rather by derivation from different ancestral prototypesof DNA-adenine methylase genes.

We appreciate the assistance of Bill Greenberg in synthesizingoligonucleotides used in this work and of Dr. John Dunn and W.Crockett in providing the facilities for such synthesis. Dr. WalterGuild generously shared his insights on restriction of plasmid trans-fer. This research was conducted by Brookhaven National Labora-tory under the auspices of the U.S. Department of Energy Office ofHealth and Environmental Research. It was supported by U.S.Public Health Service Grant GM29721.

1. Lacks, S. A., Mannarelli, B. M., Springhorn, S. S. & Greenberg,B. (1986) Cell 46, 993-1000.

2. Lacks, S. & Greenberg, B. (1975) J. Biol. Chem. 250, 4060-4066.3. Lacks, S. & Greenberg, B. (1977) J. Mol. Biol. 114, 153-168.4. Muckerman, C. C., Springhorn, S. S., Greenberg, B. & Lacks,

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Biochemistry: Cerritelli et al.