Dpna, a Methylase for Single-Strand DNA in the Dpn II Restriction System

Proc. Natl. Acad. Sci. USA Vol. 86, pp. 9223-9227, December 1989 Biochemistry DpnA, a methylase for single-strand DNA in the Dpn II restriction system, and its biological function (methyltransferase specificity/DNA sequence recognition/genetic transformation/plasmid transfer/Streptococcus pneumoniae) SUSANA CERRITELLI, SYLVIA S. SPRINGHORN, AND SANFORD A. LACKS Biology 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 by results from integration into the chromosome (9) is suscep- the Dpn II restriction gene cassette were purified, and their tible to the restriction enzymes. activities were compared on various DNA substrates. DpnA Plasmid transfer in S. pneumoniae by way of the transfor- was able to methylate single-strand DNA and double-strand mation pathway for DNA uptake is only mildly restricted by DNA, whereas DpnM methylated only double-strand DNA. the Dpn I and Dpn II systems, with transfer reduced to -40% Although both enzymes act at 5'-GATC-3' in DNA, DpnA can in the cross-transformation (7). Greater susceptibility of also methylate sequences altered in the guanine position, but at plasmid transfer than of chromosomal transformation to a lower rate. A deletion mutation in the dpnA gene was restriction would be expected from the need to reconstitute constructed and transferred to the chromosome. Transmission double-strand plasmids from the single-strand fragments that by way of the transformation pathway of methylated and enter the cell (10). Plasmid establishment presumably re- unmethylated plasmids to dpnA mutant and wild-type recipi- quires considerable new synthesis to repair gaps in the ents was examined. The mutant cells restricted unmethylated reconstituted structure (10). In the case of transfer of an donor plasmid establishment much more strongly than did unmethylated plasmid to a Dpn II-containing recipient, such wild-type cells. In the wild type, the single strands of donor synthesis would create susceptible sites. Therefore, it is plasmid DNA that enter by the transformation pathway are puzzling that the restriction effect observed on plasmid apparently methylated by DpnA prior to conversion of the transfer is so slight. plasmid to a double-strand form, in which the plasmid would The Dpn II cassette contains three genes, dpnM, dpnA, be susceptible to the Dpn II endonuclease. The biological and dpnB (1), that encode, respectively, two DNA methyl- function of DpnA may, therefore, be the enhancement of ases, DpnM and DpnA, and the Dpn II endonuclease (11). All plasmid transfer to Dpn TI-containing strains of Streptococcus three enzymes recognize 5'-GATC-3' sequences in DNA, but pneumoniae. DpnA appeared to be less constrained than the others in its specificity (3, 11). In this work, an improved procedure for The Dpn I and Dpn II restriction systems are found in purifying large amounts ofthe DpnA methylase was devised. different strains of Streptococcus pneumoniae. They are Its substrate specificity was examined and compared to that encoded by alternative genetic cassettes located at the same of the other methylase, DpnM. Furthermore, a deletion position in the chromosome (1). Dpn I is an unusual restric- mutation of dpnA was constructed and introduced into the tion endonuclease in that it only acts on methylated sites in chromosome. The effects of the mutation on restriction of DNA (2). The two systems are complementary and mutually plasmid transfer and chromosomal transformation were de- exclusive since DNA from a Dpn I strain, which is not termined. methylated at 5'-GATC-3' sites, is susceptible to Dpn II, and DNA from a Dpn II strain, which contains 5'-GmATC-3', is MATERIALS AND METHODS susceptible to Dpn I (3). Thus, when grown on the comple- Bacterial Strains and Plasmids. Strains and plasmids of S. mentary strain, bacterial viruses are reduced in infectivity to pneumoniae (1, 7, 12) and Escherichia coli (11) were previ- a level <10-5 (4). Existence of the complementary systems ously described except for those constructed in this work. may enhance survival of the species with respect to viral Growth and Transformation of Bacteria. Cultures of S. epidemics (5). The susceptibility of viruses to restriction pneumoniae were grown in semisynthetic medium (13); they results from their introduction into the cell of their double- were treated with DNA and selected for chromosomal trans- strand DNA. Dpn I and Dpn II act only on double-strand formation or plasmid establishment as described (13, 14). DNA, when both strands are methylated and unmethylated, Novobiocin-resistant (Nov9 transformants were selected respectively, at 5'-GATC-3' sites; neither enzyme acts on with novobiocin at 10 ,g/ml and chloramphenicol-resistant single-strand DNA or on hemimethylated double-strand (Cm9 transformants were selected with chloramphenicol at DNA, in which one strand is methylated and the other strand 2.5 ,ug/ml. To screen for Cm-sensitive (Cms) transformants, is not (6). we adapted a method used for maltose-negative (Mal-) clones Chromosomal transformation is not affected by the Dpn (13); with 0.2% sucrose and chloramphenicol at 1.0 ,ug/ml, restriction enzymes in the recipient cell. Cells are trans- Cms clones gave small colonies whereas Cmr clones gave formed with respect to a chromosomal marker at the same large colonies. frequency, whether or not the donor DNA is methylated, in Procedures used for the growth and transformation of E. both Dpn I- and Dpn II-containing recipients (1, 7). This lack coli and for hyperexpression ofDpn II system enzymes have of restriction effect presumably reflects the molecular fate of been reported (11). The latter depended on induction of T7 DNA in transformation: neither the single strands that enter RNA polymerase in BL21(DE3) hosts (15). the cell (8) nor the hemimethylated heteroduplex DNA that Abbreviations: Nov, novobiocin; Cm, chloramphenicol; Str, strep- The publication costs of this article were defrayed in part by page charge tomycin; Mal, maltose; Tc, tetracycline; r, resistan(t)(ce); s, sensi- payment. This article must therefore be hereby marked "advertisement" tive; ds 12-mer, 5'-CGCGGATCCGCG-3'; ss 12-mer, 5'-ATTA- in accordance with 18 U.S.C. §1734 solely to indicate this fact. GATCGCCG-3'. 9223 Downloaded by guest on October 3, 2021 9224 Biochemistry: Cerritelli et al. Proc. Natl. Acad. Sci. USA 86 (1989) Enzyme Purification and Assay. The method used for chromosomal homology to the left of the Dpn II cassette is purifying DpnM has been described (16). Details of the increased, yet the dpnA275 mutation is retained (Fig. 1). modified procedure for purifying DpnA were similar, with The dpnA275 mutation was introduced into the chromo- exceptions as noted. Methylase assays, in which [14C]methyl some of strain 1134 by transforming with pLS395 and select- groups were transferred from S-adenosylmethionine to ing for the Cms strain 1135. The proximity of the dpnA DNA, were carried out as described (17). mutation to the cat insertion site resulted in the presence of Preparation and Manipulation of DNA. Methods used for the mutation in most of the Cms transformants. To confirm preparation of plasmid and chromosomal DNA were previ- the presence of dpnA275 in the chromosome, strain 1135 was ously indicated (11). Thymus DNA was obtained commer- transformed to Tcr using Sac I-cleaved pLS402, which forced cially. M13mp8 DNA was prepared (18) from phage grown on chromosomal facilitation and transfer of the Dpn II cassette E. coli K440, a dam-3, F+ strain, kindly provided by M. to the plasmid; the resulting plasmids were examined for the Marinus (University of Massachusetts, Worcester). Oligo- presence of the additional Nhe I site. A different route was nucleotides were synthesized by the phosphoramidite used to introduce dpnA275 into the chromosome to make method on a Systec Microsyn 1450 DNA synthesizer. DNA strain 1137. First, the cat marker was transferred into pLS395 manipulations in vitro were carried out by standard methods to give pLS397. The latter then transformed 733 to Cmr (19). For a review of in vivo recombination mechanisms in S. together with dpnA275. Finally, the cat gene was removed by pneumoniae transformation and plasmid transfer, see ref. 20. transformation with pLS395 to give 1137. Although dpnA275 Construction of dpnA Mutant Strains. A deletion mutation blocked DpnA activity in methylation of single-strand DNA, of dpnA was constructed in vitro using pLS211 (Fig. 1). The the mutation had no effect on the levels of DpnM methylase plasmid was partially digested with Dra I to remove a or Dpn II endonuclease expressed by cells or plasmids 389-base-pair (bp) fragment entirely within the coding region carrying the mutant allele (data not shown). for DpnA (1). An Nhe I linker (5'-GGCTAGCC-3') was ligated into the site of the deletion to give pLS259. The mutated dpnA segment was transferred to a streptococcal RESULTS plasmid by ligating the 1.9-kilobase (kb) Nco I-BamHI frag- Purification of the DpnA Methylase. An earlier purification ment of pLS259 with the vector-containing 6.9-kb Nco I- was modified to prepare larger amounts of BamHI fragment of pLS202 (1) to give pLS275. The mutant procedure (11) allele was called dpnA275. DpnA. After hyperexpression in the BL21(DE3) (pLS211) To assist the transfer of dpnA275 to the chromosome, we system, variable and often large amounts of the Dpn proteins used a cat gene marker conferring Cmr (12), which had been in extracts were insoluble (Fig. 2a). DpnA precipitates at low previously inserted into an EcoRV site 96 bp upstream of the salt concentration, and for this protein the insoluble fraction Dpn I cassette to give pLS281 (21). On transfer of pLS281 to could contain as much as 80% of the total amount present. a Dpn II strain, 764, chromosomal facilitation of plasmid DpnA was purified, therefore, from both soluble and insol- establishment (14) substituted the Dpn II cassette down- uble fractions. A 2-liter culture yielded -1 mg of electro- stream from the cat marker, giving rise to pLS283. By phoretically pure DpnA protein. transformation the cat marker was introduced from pLS281 The soluble extract was treated with bovine DNase I and and pLS283, respectively, into the chromosomes of the Dpn passed over a column of DEAE-Sepharose CL-6B (Pharma- I strain 1133 and the Dpn II strain 1134 (illustrated in Fig. 1). cia) as described for DpnM (16). The breakthrough material The presence of the cat insert had no effect on expression of was then fractionated on heparin-agarose (Bio-Rad) to sep- Dpn I (21) or Dpn II system genes (this work; data not arate DpnA from DpnM (Fig. 2b). Further purification by shown). These derivatives enabled us to obtain plasmid agarose gel filtration on Superose 12 (Pharmacia) gave DpnA clones of Dpn I and Dpn II with greater lengths of chromo- free of other detectable proteins (Fig. 2c). somal DNA on either side of the restriction gene cassette. To The insoluble fraction, which sedimented during centrifu- accomplish this, we cloned the 7.0-kb BamHI fragment from gation at 17,000 x g for 30 min, was suspended in extraction the chromosome of strain 1133 into pLS10 (22) to give buffer containing 1 M NaCl. Most of the DpnA, but not other pLS402 (Fig. 1). Introduction of this plasmid into strain 764 proteins, dissolved, thereby resulting in partial purification of gave pLS411 by chromosomal facilitation. The 3.9-kb Pst I DpnA (Fig. 2d). After dilution to 0.3 M NaCl and chroma- fragment of pLS411 was used to transform plasmid pLS275 tography on heparin-agarose, considerable purity was to give pLS395, which rendered the cells tetracycline resist- achieved, with some fractions devoid of contaminating ant (Tcr). In pLS395 the tet gene of the vector is restored, DpnM (Fig. 2e). pLS211 pLS402 Cal T P B C P DDB PT T B

i II O ..- I----p- -I-0- amp ori PT7 dponM A B rep let dpnC D pET -5 I1kb PLSIO T pLS259 pLS41 v T P B C P NB PT B P P B TNC P DDB PT T B 1~ ~~~ . FIG. 1. Construction of a dpnA mutation and its rol -i -B 0__ _ 0 transfer to the chromosome. Plasmids are shown lin- omp ori PT7 dponM B rep let dpnM A B earized with vector as thin line and dpn chromosomal Y segment as heavy line. Chromosomal segments of pLS275 pLS395 V strains are bordered by heavy dashed line. Intact genes T N T P P C P NB PT T N T P P B TN C P NB PT are indicated with arrows showing direction of tran-

- scription. The cat gene insert was a Dpn I fragment _ moiM rep dpnM B mo/M rep tet dpnlM B from pJS3. Paths of construction are shown by block arrows: open, in vitro construction; filled, in vivo Strain Cat Strain construction by transformation. Only some restriction 1 134 j TcX D DB PT T B ,, B TN C P NB PT T B sites are shown, according to the following code: B, aB B- R- *-"M -1 10 1 1 BamHI; C, Nco I; D, Dra I; N, Nhe I; P, Pst I; S, Sac dpnM A B dpnM B I; T, BstEII. Downloaded by guest on October 3, 2021 Biochemistry: Cerritelli et al. Proc. Natl. Acad. Sci. USA 86 (1989) 9225

(a) (b) (c) (d) (e) Table 1. Comparison of DpnA and DpnM methylation of and 123 12345 2345 single-strand double-strand DNA Methyl groups transferred to DNA, pmol/hr per DNA,t ,.g of enzyme* 68 _ DNA substrate Ag DpnM DpnA Thymus DNA 16 320 97

43 ... VIW -.0- M13mp8 DNA 16 <3 870 29 _" _ ds 12-mer 1 310 260 ss 12-mer 1 15 430 -WWWY -.4- L- *Values are averages of three or more determinations using 0.03- 1 4_ 0.15 ,ug of enzyme. Amounts were chosen to give <50%16 of maximum 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, respectively. FIG. 2. Purification of the DpnA methylase. Samples of protein base of the recognition sequence. It is evident that DpnA can at various stages of purification were subjected to SDS/poly- methylate sequences other than 5'-GATC-3', when the al- acrylamide gradient gel electrophoresis (23) and stained with tered base is in the position of guanine. However, the Coomassie blue. Amounts offractions applied are expressed in terms ofequivalent volumes ofthe induced culture of BL21(DE3) (pLS211), methylating activity on these deviant substrates is only 12% which contained 0.3 mg of protein per ml. (a) Crude extract. Lane 1, or less than with the canonical site. Alteration ofbases in the protein markers (indicated by scale in other gels), 1 gg each; lane 2, thymine or cytosine positions reduces activity more com- pellet, 0.1 ml; lane 3, supernatant, 0.1 ml. (b and c) Successive steps pletely, although a little activity was observed on substitution in purification of the supernatant enzyme. (b) Heparin-agarose frac- by the other pyrimidine. tions. Lanes 1-5, even fractions 34-42, 2 ml. (c) Superose gel filtration In vivo DpnA methylates DNA at the N6 position of fraction 53, 4 ml. (d) Pellet dissolved in 1 M NaCl, 1 ml. (e) adenine in 5'-GATC-3', as shown by susceptibility of the Heparin-agarose fractions of dissolved pellet. Lanes 1-5, even fractions 22-30, 2 ml. Arrows indicate positions expected for DpnA methylated DNA to Dpn I. The methylation of related (upper) and DpnM (lower). sequences in vitro also appears to be at this position. No methylation was observed with oligonucleotides substituted Action of DpnA on Single-Strand DNA. The methylating in the adenine position by either guanine, thymine, or cy- activity of purified DpnM and DpnA was compared with a tosine (data not shown). Substitution of cytosine, the only variety ofDNA substrates unmethylated at 5'-GATC-3' sites. other potentially methylatable base in the recognition se- These included double-strand DNA from calfthymus, single- quence, by thymine did not completely eliminate methylation strand DNA from phage M13mp8, and synthetic oligodeoxy- (Table 2). nucleotides 5'-CGCGGATCCGCG-3' (ds 12-mer) and 5'- Effect of dpnA on Restriction of Plasmid Establishment. ATTAGATCGCCG-3' (ss 12-mer). The first oligonucleotide Isogenic pairs of strains carrying the wild-type and dpnA275 is self-complementary, and =90% was in a double-strand mutant Dpn II gene cassettes in their chromosomes were form after gel electrophoresis; the second appeared to be only compared with respect to restriction of plasmid establish- in a single-strand form (results not shown). ment and chromosomal transformation. The plasmids used- Both methylases are active with thymus DNA, although pJS3, pMV158, and pLS70-all contain the pLS1 replicon DpnM shows a somewhat higher specific activity (Table 1). (24) and, respectively, 2, 8, and 11 5'-GATC-3' sites (12, 25). Most strikingly, however, DpnA is highly active in methyl- These plasmids, as well as chromosomal DNA, were pre- ating the single-strand M13 DNA, even more than with pared in unmethylated and methylated form from Dpn I- and thymus DNA. In contrast, DpnM showed no detectable Dpn II-containing hosts, respectively. Transformation was activity on M13mp8 DNA. Consistent with the specificity of carried out with -0.1 ,ug of plasmid DNA (except for DpnA for single-strand and double-strand DNA is its high unmethylated pLS70, for which about halfthis concentration activity on both oligonucleotides. DpnM showed high activ- was used) or 1.0 ,ug of chromosomal DNA per ml of culture. with the double-strand With the ity oligonucleotide. "single- Table 2. Nucleotide sequence specificity for methylation strand" oligonucleotide, however, DpnM showed about 5% by DpnA ofits activity on the double-strand oligonucleotide. Inasmuch as the four nucleotides constituting the recognition site are Oligodeoxynucleotide pmol methylated Relative complementary, they may provide sufficient double- substrate* per hr/,ug of DpnA action strandedness to the ss 12-mer to allow some action by DpnM. 5'-ATTAGATCGCCG-3' 810 (1.00) Conversely, the apparently higher specific activity of DpnA 5'-ATIAAAMGCCG-3' 100 0.12 on the ds 12-mer than on thymus DNA may result from a 5'-ATrACAICGCCG-3' 46 0.06 significant amount (10%) of the former remaining in a single- 5'-ATTATACGCCG-3' 29 0.04 strand form. 5'-ATTAQACCGCCG-3' 15 0.02 Specificity of DpnA Methylation in Vitro. Earlier studies 5'-ATTAQAACGCCG-3' <5 <0.01 showed that DpnA methylated 5'-GATC-3' sites in vivo, but 5'-ATTAUAGCGCCG-3' <5 <0.01 in vitro the enzyme methylated other sites, as shown by its 5'-ATrAQAUIGCCG-3' 6 0.01 ability to methylate DNA already methylated at 5'-GATC-3' 5'-ATTAjATGGCCG-3' <5 <0.01 sites (11). We therefore examined activity of the purified 5'-ATfAQAIAGCCG-3' <5 <0.01 enzyme on a variety of oligonucleotide substrates containing Recognition sequence is underlined; altered bases are shown in 5'-GATC-3' and related sequences. Table 2 presents results boldface. with a set of single-strand oligonucleotides varying in a single *Five micrograms (1250 pmol) in assay mixture. Downloaded by guest on October 3, 2021 9226 Biochemistry: Cerritelli et al. Proc. Natl. Acad. Sci. USA 86 (1989) Comparison with results for transformation of the null re- Results for the dpnA275 recipient strains are markedly striction strain, 762, affords a correction for variation in different (Table 4). Establishment of unmethylated plasmids quality or quantity of the donor DNA. Selection for Cmr or is restricted to a level between 0.2% and 7% of methylated Tcr measured plasmid establishment; selection for Mal', plasmid establishment. The extent of restriction correlates streptomycin resistance (Str9), or Novr measured chromo- with the number ofDpn II restriction sites in the plasmids: 2 somal transformation. for pJS3, 8 for pMV158, and 11 for pLS70. Plasmids without One pair of recipient strains was isogenic with strain 762. homology to the chromosome, such as pJS3 and pMV158, are These are 764, in which the Dpn II cassette replaced the established only by complementary plasmid strand reassem- defective Dpn I cassette of762 (1, 4), and its derivative, strain bly (10, 14). However, inasmuch as pLS70 contains a large 1135. The other pair, 733 and 1137, are derivatives of the segment of DNA homologous to the recipient chromosome, original S. pneumoniae strain from which the Dpn II cassette most of its establishment must occur by chromosomal facil- was obtained. All four recipients contain malM point muta- itation rather than by direct plasmid reconstitution (14). tions that allow measurement of chromosomal transforma- Thus, restriction by Dpn II in dpnA mutants, which strongly tion by the malMgene cloned in pLS70. The data obtained for reduces transfer ofboth pMV158 and pLS70, must affect both strain 762 and both isogenic pairs (with wild-type or mutant processes of plasmid establishment. dpnA) are shown in Table 3. To calculate the restriction A most plausible explanation for the severe restriction of effect, the ratio of transformants with unmethylated relative plasmid establishment in dpnA mutants, but not in wild-type to donor DNA was divided recipients, is that the methylation of incoming single-strand methylated by the ratio given by DNA by the DpnA methylase protects the plasmid sites from the null strain. These results are presented in Table 4. The later attack by the Dpn II endonuclease. In the absence of results with the wild-type recipients, 764 and 733, confirm such methylation, attack by Dpn II would follow conversion earlier work (7) by showing no restriction of chromosomal ofthe plasmid DNA to a double-strand unmethylated form by transformation and only limited restriction, -50%, of plas- (i) association with a complementary donor plasmid strand, mid establishment. (ii) repair synthesis ofunpaired segments after partial plasmid Table 3. Effect of dpnA mutation on restriction of reconstitution, or (iii) complementary strand synthesis after plasmid transfer circular synapsis (20) of a plasmid strand with chromosomal DNA. The primary biological function of the DpnA methyl- Transformants x Dpn I ase may be to allow the transfer of plasmids from Dpn I 10-3/mlt donor/ strains of S. pneumoniae or from other bacterial species to Recipient Donor Dpn I Dpn II Dpn II Dpn II strains. strain* DNA Marker straint strain§ donor Some restriction of chromosomal transformation by unmethylated DNA may also occur in dpnA275 recipients 762 pJS3 Cmr 1.3 0.80 1.62 (Table 3). This was not evident for the Mal' marker carried (null) pMV158 Tcr 2.8 2.6 1.08 by plasmid pLS70, but the two chromosomal resistance pLS70 Tcr 6.1 17 0.36 markers showed restriction effects to the level of 20-50%. pLS70 Mal' 390 1,200 0.33 Chrom. Strr 90 100 0.90 Unless Dpn II can act to some extent on single-strand DNA, Chrom. Novr 130 140 0.93 which has not been observed (6), an explanation for this 764 pJS3 Cmr 0.73 0.75 0.97 effect is not apparent. However, to the extent that such (wild) pMV158 Tcr 1.1 1.6 0.69 restriction occurs, the DpnA methylase would enhance, also, pLS70 Tcr 7.9 34 0.23 chromosomal transformation. pLS70 Mal' 350 1,100 0.32 Chrom. Strr 42 50 0.84 DISCUSSION Chrom. Novr 57 82 0.70 The DpnA methylase is unusual in its ability to methylate 1135 pJS3 Cmr 0.33 3.0 0.11 single-strand DNA. It appears to have evolved for this (A275) pMV158 Tcr 0.20 5.4 0.037 particular function inasmuch as the Dpn II restriction system, pLS70 Tcr 0.30 72 0.0042 a Mal' 830 0.69 of which it is part, already contains a potent methylase for pLS70 1,200 double-strand DNA, DpnM. Its primary biological role may Chrom. Strr 84 530 0.16 be to enable plasmid transmission to Dpn II-containing cells Chrom. Novr 110 640 0.17 by way of the transformation pathway of DNA entry, which 733 pJS3 Cmr 0.57 0.74 0.77 DNA in the (wild) pMV158 Tcr 4.9 10 0.49 introduces single-strand form (8). Methylation of pLS70 Tcr 5.9 160 0.37 incoming strand would protect the subsequently reconsti- Mal' 0.62 tuted plasmid from Dpn II cleavage. The mechanism of pLS70 5,600 9,100 chromosomal transformation itself assures its resistance to Chrom. Strr 250 260 0.96 restriction (6). Thus, the systems ofS. pneumoniae that allow Chrom. Novr 260 210 1.24 the species to benefit from genetic exchange and plasmid 1137 pJS3 Cmr 2.4 28 0.086 transfer are largely immune to restriction, which is presum- (A275) pMV158 Tcr 0.29 35 0.0083 ably directed at the prevention of bacterial virus infection. pLS70 Tcr 0.23 380 0.00061 Earlier work showed that methylated plasmids were little pLS70 Mal' 20,000 49,000 0.41 restricted in Dpn I-containing cells. This can be explained by Chrom. Strr 320 760 0.42 the resistance to Dpn I cleavage of reconstituted plasmids Chrom. Novr 480 960 0.50 containing large regions of newly synthesized strands, which Chrom., chromosomal DNA. would be unmethylated (10). A similar explanation was *Dpn II genotype is shown in parentheses; strain pairs 764 and 1135 untenable for the mild restriction ofunmethylated plasmids in and 733 and 1137 are isogenic. Dpn II-containing cells (7). As indicated above, however, this tNumber of transformants per ml with donor DNA from Dpn I or mild restriction can now be attributed to methylation by Dpn II strain. tPlasmid host was 217; chromosomal DNA from 533; DNA was DpnA of the single-strand donor DNA that enters the cell by unmethylated. way of the transformation pathway. §Plasmid host was 697; chromosomal DNA from 1138, nov trans- Previously observed differences between restriction of formant of 697; DNA was methylated. plasmid transfer in the conjugative and transformation path- Downloaded by guest on October 3, 2021 Biochemistry: Cerritelli et al. Proc. Natl. Acad. Sci. USA 86 (1989) 9227 Table 4. Summary of restriction effects on plasmid transfer Restriction 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 0.11 pMV158 Tcr 0.64 0.034 0.053 0.45 0.008 0.017 pLS70 Tcr 0.64 0.012 0.019 1.03 0.002 0.002 pLS70 Mal+ 0.97 2.1 2.2 1.88 1.24 0.66 Chrom. Stif 0.75 0.18 0.24 1.07 0.47 0.44 Chrom. 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 methylated donor 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 latter We appreciate the assistance of Bill Greenberg in synthesizing case. On transfer from a Dpn II strain to a Dpn I strain, the oligonucleotides used in this work and of Dr. John Dunn and W. Crockett in providing the facilities for such synthesis. Dr. Walter conjugative plasmid pIP501 was not restricted (W. Guild, Guild generously shared his insights on restriction of plasmid trans- personal communication), which is consistent with conjuga- fer. This research was conducted by Brookhaven National Labora- tive transfer of only a single strand. As in the transformation tory under the auspices of the U.S. Department of Energy Office of pathway, replication ofthe complementary strand would give Health and Environmental Research. It was supported by U.S. a hemimethylated duplex, which is not a substrate for Dpn I Public Health Service Grant GM29721. (6). In contrast, unmethylated pIP501 was restricted to 1i-4 1. Lacks, S. A., Mannarelli, B. M., Springhorn, S. S. & Greenberg, when transferred to a wild-type Dpn II strain (26). The B. (1986) Cell 46, 993-1000. smaller plasmid pMV158, when mobilized by pIP501, was 2. Lacks, S. & Greenberg, B. (1975) J. Biol. Chem. 250, 4060-4066. restricted to 2%. In the case of conjugative single-strand 3. Lacks, S. & Greenberg, B. (1977) J. Mol. Biol. 114, 153-168. transfer, very rapid synthesis ofthe complementary strand by 4. Muckerman, C. C., Springhorn, S. S., Greenberg, B. & Lacks, S. A. (1982) J. Bacteriol. 152, 183-190. enzymes introduced with the DNA during conjugation, which 5. Lacks, S. A., Mannarelli, B. M., Springhorn, S. S., Greenberg, B. can occur during conjugative transfer (27), presumably ren- & de la Campa, A. G. (1987) in Streptococcal Genetics, eds. ders the DNA susceptible to the Dpn II endonuclease prior Ferretti, J. J. & Curtiss, R., III (Am. Soc. Microbiol., Washington, In DC), pp. 31-41. to action of DpnA. transformation, the less rapid recon- 6. Vovis, G. F. & Lacks, S. (1977) J. Mol. Biol. 115, 525-538. stitution and repair synthesis dependent on host enzymes 7. Lacks, S. A. & Springhorn, S. S. (1984) J. Bacteriol. 158, 905-909. would allow time for DpnA action. 8. Lacks, S. (1962) J. Mol. Biol. 5, 119-131. In addition to acting on single-strand DNA, DpnA differs 9. Fox, M. S. & Allen, M. K. (1964) Proc. Natl. Acad. Sci. USA 52, 412-419. from DpnM in its less stringent requirement for the 5'- 10. Saunders, C. W. & Guild, W. R. (1980) Mol. Gen. Genet. 181, GATC-3' target sequence. Recognition of adenine, thymine, - 57-62. and cytosine was critical for action, but variation of the first 11. de la Campa, A. G., Kale, P., Springhorn, S. S. & Lacks, S. A. base, guanine, was tolerated to some extent. The signifi- (1987) J. Mol. Biol. 196, 457-469. 12. Ballester, S., Lopez, P., Alonso, J. C., Espinosa, M. & Lacks, cance, if any, of this degeneracy is unknown. S. A. (1986) Gene 41, 153-163. Also unexplained at present is the apparent redundancy of 13. Lacks, S. (1966) Genetics 53, 207-235. DpnM, inasmuch as DpnA itself can methylate 5'-GATC-3' 14. Lopez, P., Espinosa, M., Stassi, D. L. & Lacks, S. A. (1982) J. in double-strand DNA. When the Dpn II system cassette is Bacteriol. 150, 692-701. present on a multicopy plasmid containing intact dpnA and 15. Studier, F. W. & Moffatt, B. A. (1986) J. Mol. Biol. 189, 113-130. 16. Cerritelli, S., White, S. W. & Lacks, S. A. (1989) J. Mol. Biol. 208, dpnB, the dpnM gene is not essential (11). It appears that a 841-842. multiple dose of the dpnA gene expresses sufficient DpnA 17. Lacks, S. A. & Springhorn, S. S. (1984) J. Bacteriol. 157, 934-936. methylase to protect cellular DNA from the Dpn II endonu- 18. Yamamoto, K. R., Alberts, B. M., Benzinger, R., Lonhorne, L. & clease. Further investigation is necessary to determine Treiber, G. (1970) Virology 40, 734-740. whether a single copy of dpnA in the chromosome will also 19. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold allow the cell to dispense with DpnM. Spring Harbor, NY). The amino acid sequence of DpnA is distinct from DpnM 20. Lacks, S. A. (1988) in Genetic Recombination, eds. Kucherlapati, (1); only three small boxes of similarity, common to a variety R. & Smith, G. (Am. Soc. Microbiol., Washington, DC), pp. 43-85. of DNA adenine methylases (11, 28), are evident. The 21. de la Campa, A. G., Springhorn, S. S., Kale, P. & Lacks, S. A. sequence of the Hinfl methylase ofHaemophilus influenzae, (1988) J. Biol. Chem. 263, 14696-14702. 22. Puyet, A., Greenberg, B. & Lacks, S. A. (1989) J. Bacteriol. 171, which methylates adenine in 5'-GANTC-3', was recently 2278-2286. reported (29). DpnA is very similar to this protein; 40% of its 23. Weinrauch, Y. & Lacks, S. A. (1981) Mol. Gen. Genet. 183, 7-12. residues are identical to the 255 amino-terminal residues of 24. Lacks, S. A., Lopez, P., Greenberg, B. & Espinosa, M. (1986) J. the Hinfl methylase. The two proteins are presumably ho- Mol. Biol. 192, 753-765. mologous in a biological sense-that is, descended from a 25. Lacks, S. A., Dunn, J. J. & Greenberg, B. (1982) Cell 31, 327-336. 26. Guild, W. R., Smith, M. D. & Shoemaker, N. B. (1982) in Micro- common ancestral form. However, DpnA does not methylate biology 1982, ed. Schlessinger, D. (Am. Soc. Microbiol., Washing- 5'-GANTC-3' sequences in oligonucleotides, with the excep- ton, DC), pp. 82-92. tion of5'-GAATC-3' (data not shown). DpnM was previously 27. Willetts, N. & Wilkins, B. (1984) Microbiol. Rev. 48, 24-41. found to be homologous to the Dam methylase ofE. coli (30). 28. Lauster, R. (1989) J. Mol. Biol. 206, 313-321. Despite their adjacent location in the Dpn II gene cassette, it 29. Chandrasegaran, S., Lunnen, K. D., Smith, H. 0. & Wilson, G. G. (1988) Gene 70, 387-392. appears that dpnA and dpnMdid not arise by gene duplication 30. Mannarelli, B. M., Balganesh, T. S., Greenberg, B., Springhorn, but rather by derivation from different ancestral prototypes S. S. & Lacks, S. A. (1985) Proc. Natl. Acad. Sci. USA 82, of DNA-adenine methylase genes. 4468-4472. Downloaded by guest on October 3, 2021