5208–5221 Nucleic Acids Research, 2009, Vol. 37, No. 15 Published online 3 July 2009 doi:10.1093/nar/gkp534 The MmeI family: type II restriction–modification enzymes that employ single-strand modification for host protection Richard D. Morgan*, Elizabeth A. Dwinell, Tanya K. Bhatia, Elizabeth M. Lang and Yvette A. Luyten New England Biolabs, Inc. 240 County Road Ipswich, MA 01938 USA Received March 18, 2009; Revised and Accepted June 8, 2009 ABSTRACT material between ‘self’ and ‘others’ within microbial popu- lations (2). As such they represent fertile ground for the The type II restriction endonucleases form one of study of protein evolution, particularly the evolution the largest families of biochemically-characterized of those protein–DNA interactions that confer sequence proteins. These endonucleases typically share little specificity to these enzymes. sequence similarity, except among isoschizomers A restriction system must differentiate between ‘self’ that recognize the same sequence. MmeI is an unu- and ‘foreign’ DNA in order to cut the identifiably foreign sual type II restriction endonuclease that combines DNA but not host DNA. This discrimination is based endonuclease and methyltransferase activities in a upon modification of the DNA, usually in the form of single polypeptide. MmeI cuts DNA 20 bases from methylation of a base in each strand within the discrete its recognition sequence and modifies just one DNA DNA sequence recognized by the endonuclease. Usually, strand for host protection. Using MmeI as query we hemi-methylated DNA is not cut by the type II endonu- have identified numerous putative genes highly sim- cleases, ensuring that immediately following replication the hemi-methylated daughter chromosomes remain pro- ilar to MmeI in database sequences. We have cloned tected from cleavage by the endonuclease, allowing the and characterized 20 of these MmeI homologs. DNA methyltransferase time to modify the newly repli- Each cuts DNA at the same distance as MmeI and cated strand. Type II R–M systems most commonly have each modifies a conserved adenine on only one DNA separate enzymes to accomplish host DNA modification strand for host protection. However each enzyme and endonuclease cleavage of foreign DNA. However, recognizes a unique DNA sequence, suggesting some systems, such as some members of the type IIG these enzymes are undergoing rapid evolution of subgroup, have one polypeptide that contains both DNA specificity. The MmeI family thus provides a DNA methyltransferase and endonuclease activity, part- rich source of novel endonucleases while affording nered with a second, companion DNA methyltransferase an opportunity to observe the evolution of DNA (3,4). The type IIG enzymes generally recognize asymmet- specificity. Because the MmeI family enzymes ric sequences and cut away from the recognition sequence. employ modification of only one DNA strand for The fusion proteins cut fully unmodified DNA, but can host protection, unlike previously described type II also modify a base in one DNA strand of unmodified DNA. Because the recognition site is not disrupted by systems, we propose that such single-strand the endonucleolytic cleavage, they can remain bound to modification systems be classified as a new sub- and modify their recognition site even after cutting the group, the type IIL enzymes, for Lone strand DNA DNA. However, previously described type IIG endonu- modification. cleases, such as Eco57I, require a separate, companion DNA methyltransferase in order to achieve modification of both DNA strands, since the single-strand modification INTRODUCTION produced by the fusion protein is insufficient for host The type II restriction endonucleases and methyltrans- protection (5). ferases constitute a large and widely distributed family Since most bacteria and archaea possess one or more of enzymes (1). These enzymes are under strong selective restriction systems, the recent availability of complete pressure as a primary defense against parasitic DNA. genome sequences has provided a wealth of putative They may also function to control the exchange of genetic restriction systems. These are typically identified in silico *To whom correspondence should be addressed. Tel: 978 927 5054; Fax: 978 921 1350; Email: [email protected] ß 2009 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2009, Vol. 37, No. 15 5209 through amino-acid sequence similarity to conserved Endonuclease assays sequence motifs within the DNA methyltransferase. The Endonuclease activity was assayed by incubating various type II DNA methyltransferases exhibit significant amino- amounts of enzyme in reaction buffer (NEBuffer 4: 20 mM acid sequence similarity in the regions involved in binding Tris–acetate, pH 7.9, 10 mM magnesium acetate, 50 mM the methyl donor AdoMet and the catalysis of methyl potassium acetate, 1 mM DTT, supplemented with 100 mg/ transfer, which allows uncharacterized putative sequences ml BSA and 80 mM AdoMet) containing 1 mg substrate to be reliably identified as DNA methyltransferases (6,7). DNA per 50 ml for one hour at 378C. Reactions were ter- However, the type II restriction endonucleases (REs) minated by addition of stop solution (50 mM EDTA, pH are generally not similar at the level of primary amino- 8.0, 50% glycerol, 0.02% bromophenol blue), and reac- acid sequence, even though many share a conserved struc- tion products were analyzed by electrophoresis in 1% ture (8). Type II REs typically exhibit a low level of LE agarose gels alongside DNA size standards lambda- sequence identity that falls in the so-called ‘twilight HindIII and PhiX174-HaeIII, or lambda-BstEII and zone’ of sequence similarity, where genuine similarities pBR322-MspI. between homologs disappear into the random noise of sequence comparison (9,10). Although significant similar- Identification of MmeI homologs in sequence databases ity between putative and characterized endonucleases is occasionally observed, such enzymes are usually isoschi- The MmeI protein sequence (GenBank accession no zomers, i.e. they recognize the same DNA sequence and ACC85607) served as the query to identify significantly cut at the same position. For most type II REs, however, similar protein sequences using BLAST searches per- the lack of sequence conservation makes identification formed against available databases, such as the non- of these enzymes among the many putative sequences redundant protein sequences (nr) or the environmental available in sequenced genomes a challenging task (11). samples (env-nr) amino-acid sequence databases at the It was therefore surprising that after cloning the unu- National Center for Biotechnology Information (NCBI), sual type II endonuclease MmeI (12), we observed a the REBASE database of restriction systems (1), or number of highly similar putative sequences in available TBLASTN searches against the Nucleotide collection databases. Expression and characterization of these (nr/nt) or environmental samples (env-nt) DNA databases MmeI-like putative genes has allowed us to describe a at NCBI (13). Significant hits with an expectation value new family of type II restriction enzymes. These enzymes <e–20 were considered potential MmeI homologs. The employ the unusual strategy of using only single-strand putative homologs were evaluated to identify those methylation for host protection, a property not previously sequences that contained the conserved PD-(D/E)xK described for type II R–M systems. Modification of only endonuclease motif residues, aligned with the putative one DNA strand raises the question of how host protec- MmeI catalytic residues D70,E80 and K82 in the amino tion is maintained, since immediately following replication terminal region, and that exhibited similarity throughout one of the two daughter chromosomes will be fully unmo- the entire length of MmeI. A number of the identified dified and would be expected to be vulnerable to restric- putative MmeI homologs were cloned and expressed in tion. However the MmeI-like systems described appear Escherichia coli. frequently in sequenced bacterial genomes, and it appears from bioinformatic analyses that such single-strand mod- Cloning and expression of MmeI homologs ification for host protection may be widespread in nature. The microbial strain, or the genomic DNA from the The diversity of recognition sequences found in this clo- strain, encoding a MmeI homolog of interest was obtained sely related family of proteins affords a unique window from the source listed in Table 1. Where purified genomic into the evolution of specific protein–DNA-binding DNA was not available, DNA was prepared from a interactions. freshly grown cell culture by standard techniques or directly from a lyophilized ATCC or DSM culture vial by chemical lysis and bead beating (MoBio MATERIALS AND METHODS Laboratories, CA). For environmental sequences, where neither genomic DNA nor the microbial strain was avail- Restriction endonucleases, S-adenosyl-L-methionine able, the putative endonuclease gene was obtained by (AdoMet), T4 DNA Ligase, DNA polymerases, DNA in vitro synthesis. size standards and competent cells were from New Putative genes were PCR amplified from genomic England Biolabs (Ipswich, MA). Synthetic DNA oligonu- DNA. The oligonucleotide