Interaction of AluI, Cfr6I and PvuII restriction-modification enzymes with substrates containing either N4-methylcytosine or 5-methylcytosine

The cleavage specificity of R.Cfr6I, an isoschizomer of PvuII restriction endonuclease was determined to be 5'CAG decreases CTG and the methylation specificity of Cfr6I and PvuII methylases, 5'CAG4mCTG. Thus, M.Cfr6I and M.PvuII are new additions to the list of methylases with N4-methylcytosine specificity. Neither of the above RM enzymes acts on the substrates containing either N4-methylcytosine or 5-methylcytosine in a cognate methylation position.     

Specificity of action of DNA methylases BcnI, CfrI and Cfr10I

The site specificity of three DNA methylases BcnI, CfrI and Cfr10I was determined to be 5'Cm4C(C/G)GG, 5'PyGGm5CCPu and 5'Pum5CCGGPy, respectively. Using the modification methylases under investigation with known restriction endonucleases, fourteen new DNA cleavage specificities can be created. Some aspects of the use of restriction endonucleases in DNA methylation analysis are discussed.     

Specificity of new restrictases and methylases. Unusual modification of cytosine at position 4

Fourteen restriction endonucleases and 4 methylases were isolated and purified from 14 strains of Citrobacter freundii and Escherichia coli, which were isolated from natural sources. To determine the nucleotide sequence recognized by the endonucleases a comparison of DNA cleavage patterns, the evaluation of the cleavage frequency of some DNA with known recognition sequences and mapping was used. It was determined that Cfr101 is a new enzyme recognizing 5'PuCCGGPy. Other restriction enzymes isolated were isoschizomers of: Cfr5I, Cfr11I, Eco60I, Eco61I--EcoRII; Cfr4I, Cfr8I, Cfr13I--Sau96I; Cfr6I--PvuII, Cfr9I--SmaI, Eco26I--HgiJII; Eco32I--EcoRV; Eco52I--XmaIII; Eco56I--NaeI. Some of the enzymes in C. freundii and E. coli were found for the first time. The methylases MCfrI; MCfr6I, MCfr9I and MCfr10I recognize the same nucleotide sequence as specific endonucleases isolated from the same strain. DNA modification in vitro by MCfrI and MCfr10I yields 5-methylcytosine and 4-methylcytosine by MCfr6I and MCfr9I.     

The emerging methicillin-resistant Staphylococcus aureus ST398 clone can easily be typed using the Cfr9I SmaI-neoschizomer

Aim:  To establish a PFGE protocol using Cfr9I, neoschizomer of SmaI, for typing of Staphylococcus aureus isolates belonging to the emerging MRSA ST398 clone.
Methods and Results:  Staphylococcus aureus ST398 and non-ST398 isolates were analysed using the PFGE conditions recommended by the HARMONY consensus protocol. Genomic DNA of non-ST398 isolates could be digested with SmaI, XmaI (also a SmaI-neoschizomer) and Cfr9I. The DNA of SmaI-nontypeable ST398 isolates was partially resistant to XmaI, but could be digested with Cfr9I. By PCR-amplification/sequencing, the presence of a novel C5-cytosine methyltransferase gene ( sauST398M ) was detected in the ST398 isolates. The encoded enzyme, which shows high similarity with C5-cytosine methyltransferases that modify the CCCGGG recognition sequence, could be responsible for the different restriction results.
Conclusion:  SmaI-PFGE is regarded as the 'gold standard' for typing S. aureus . Because of different susceptibility of the GGGCCC recognition sites of the ST398 DNA against SmaI, XmaI and Cfr9I, the proposed protocol is a valuable tool for ST398 typing.
Significance and Impact of the Study:  The use of this protocol allows the comparison of results from SmaI-nontypeable isolates with S. aureus SmaI-PFGE databases and can be applied for outbreak investigations and traceability studies of this emerging MRSA clone.     

The Cfr10I restriction enzyme is functional as a tetramer.

It is thought that most of the type II restriction endonucleases interact with DNA as homodimers. Cfr10I is a typical type II restriction enzyme that recognises the 5'-Pu decreases CCGGPy sequence and cleaves it as indicated by the arrow. Gel-filtration and analytical ultracentrifugation data presented here indicate that Cfr10I is a homotetramer in isolation. The only SfiI restriction enzyme that recognises the long interrupted recognition sequence 5'-GGCCNNNNNGGCC has been previously reported to operate as a tetramer however, its structure is unknown. Analysis of Cfr10I crystals revealed that a single molecule in the asymmetric unit is repeated by D2 symmetry to form a tetramer. To determine whether the packing of the Cfr10I in the crystal reflects the quaternary structure of the protein in solution, the tryptophan W220 residue located at the putative dimer-dimer interface was mutated to alanine, and the structural and functional consequences of the substitution were analysed. Equilibrium sedimentation experiments revealed that, in contrast to the wild-type Cfr10I, the W220A mutant exists in solution predominantly as a dimer. In addition, the tetramer seems to be a catalytically important form of Cfr10I, since the DNA cleavage activity of the W220A mutant is < 0.1% of that of the wild-type enzyme. Further, analysis of plasmid DNA cleavage suggests that the Cfr10I tetramer is able to interact with two copies of the recognition sequence, located on the same DNA molecule. Indeed, electron microscopy studies demonstrated that two distant recognition sites are brought together through the DNA looping induced by the simultaneous binding of the Cfr10I tetramer to both sites. These data are consistent with the tetramer being a functionally important form of Cfr10I.     

Specificity changes in the evolution of type II restriction endonucleases: a biochemical and bioinformatic analysis of restriction enzymes that recognize unrelated sequences

How restriction enzymes with their different specificities and mode of cleavage evolved has been a long standing question in evolutionary biology. We have recently shown that several Type II restriction endonucleases, namely SsoII (downward arrow CCNGG), PspGI (downward arrow CCWGG), Eco-RII (downward arrow CCWGG), NgoMIV (G downward arrow CCGGC), and Cfr10I (R downward arrow CCGGY), which recognize similar DNA sequences (as indicated, where the downward arrows denote cleavage position), share limited sequence similarity over an interrupted stretch of approximately 70 amino acid residues with MboI, a Type II restriction endonuclease from Moraxella bovis (Pingoud, V., Conzelmann, C., Kinzebach, S., Sudina, A., Metelev, V., Kubareva, E., Bujnicki, J. M., Lurz, R., Luder, G., Xu, S. Y., and Pingoud, A. (2003) J. Mol. Biol. 329, 913-929). Nevertheless, MboI has a dissimilar DNA specificity (downward arrow GATC) compared with these enzymes. In this study, we characterize MboI in detail to determine whether it utilizes a mechanism of DNA recognition similar to SsoII, PspGI, EcoRII, NgoMIV, and Cfr10I. Mutational analyses and photocross-linking experiments demonstrate that MboI exploits the stretch of approximately 70 amino acids for DNA recognition and cleavage. It is therefore likely that MboI shares a common evolutionary origin with SsoII, PspGI, EcoRII, NgoMIV, and Cfr10I. This is the first example of a relatively close evolutionary link between Type II restriction enzymes of widely different specificities.     

PspGI,a type II restriction endonuclease from the extreme thermophile Pyrococcus sp.: structural and functional studies to investigate an evolutionary relationship with several mesophilic restriction enzymes

We present here the first detailed biochemical analysis of an archaeal restriction enzyme. PspGI shows sequence similarity to SsoII, EcoRII, NgoMIV and Cfr10I, which recognize related DNA sequences. We demonstrate here that PspGI, like SsoII and unlike EcoRII or NgoMIV and Cfr10I, interacts with and cleaves DNA as a homodimer and is not stimulated by simultaneous binding to two recognition sites. PspGI and SsoII differ in their basic biochemical properties, viz. stability against chemical denaturation and proteolytic digestion, DNA binding and the pH, MgCl(2) and salt-dependence of their DNA cleavage activity. In contrast, the results of mutational analyses and cross-link experiments show that PspGI and SsoII have a very similar DNA binding site and catalytic center as NgoMIV and Cfr10I (whose crystal structures are known), and presumably also as EcoRII, in spite of the fact that these enzymes, which all recognize variants of the sequence -/CC-GG- (/ denotes the site of cleavage), are representatives of different subgroups of type II restriction endonucleases. A sequence comparison of all known restriction endonuclease sequences, furthermore, suggests that several enzymes recognizing other DNA sequences also share amino acid sequence similarities with PspGI, SsoII and EcoRII in the region of the presumptive active site. These results are discussed in an evolutionary context.     

Fluorometric identification of 5-methylcytosine modification in DNA: combination of photosensitized oxidation and invasive cleavage

An efficient fluorometric detection system of DNA methylation has been developed by a combination of a photooxidative DNA cleavage reaction with 2-methyl-1,4-naphthoquinone (NQ) chromophore and an invasive cleavage reaction with human Flap endonuclease-1. Enzymatic treatment of a mixture of photochemically fragmented target oligodeoxynucleotides (ODNs) at 5-methylcytosine mC) and hairpin-like probe oligomer possessing a fluorophore (F) and a quencher (D) resulted in a dramatic enhancement of fluorescence. In contrast, fluorescence emission for the ODN containing cytosine but not mC at the target sequence was extremely weak. In addition, by monitoring the fluorescence change, this system allows for the detection of mC in DNA at subfemtomole amounts. This system would provide a highly sensitive protocol for determining the methylation status in DNA by fluorescence emission.     

Circular permutation of DNA cytosine-N4 methyltransferases: in vivo coexistence in the BcnI system and in vitro probing by hybrid formation

Sequence analysis of the BcnI restriction-modification system from Bacillus centrosporus revealed four open reading frames (bcnIC, bcnIR, bcnIB and bcnIA) that are arranged as two converging collinear pairs. One pair encodes a putative small regulatory protein, C.BcnI, and the restriction endonuclease R.BcnI. The other two gene products are the DNA cytosine-N4 methyltransferases M.BcnIA and M.BcnIB, which differ by circular permutation of conserved sequence motifs. The BcnI methyltransferases are isospecific on double-stranded DNA [methylation specificity CC(C/G)GG], but M.BcnIA can also methylate the target sites in single-stranded DNA. Functional analysis shows that bcnIA is dispensable (bcnIB is capable of protecting the DNA against the in vivo activity of bcnIR); in contrast, no stable clones were obtained if bcnIB alone was deleted from the system. By analogy with the DpnII system, the second methylase M.BcnIA may play a role in the transformation proficiency of its gram-positive host. The interchangeability of homologous elements in the beta class of cytosine-N4 methylases was probed by hybrid formation between M.BcnIB and its closest homolog M.Cfr9I (CCCGGG) employing a novel semi-random strategy combined with selection for catalytic activity. The fusion points in the active hybrids mapped in a narrow region located between sequence motifs X and I. Our data illustrate that recombination of two related sequences by circular permutation may serve as an evolutionary mechanism for creating new specificities of amino MTases.     

Modification-dependent restriction endonuclease,MspJI, flips 5-methylcytosine out of the DNA helix

MspJI belongs to a family of restriction enzymes that cleave DNA containing 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC). MspJI is specific for the sequence 5(h)mC-N-N-G or A and cleaves with some variability 9/13 nucleotides downstream. Earlier, we reported the crystal structure of MspJI without DNA and proposed how it might recognize this sequence and catalyze cleavage. Here we report its co-crystal structure with a 27-base pair oligonucleotide containing 5mC. This structure confirms that MspJI acts as a homotetramer and that the modified cytosine is flipped from the DNA helix into an SRA-like-binding pocket. We expected the structure to reveal two DNA molecules bound specifically to the tetramer and engaged with the enzyme''s two DNA-cleavage sites. A coincidence of crystal packing precluded this organization, however. We found that each DNA molecule interacted with two adjacent tetramers, binding one specifically and the other non-specifically. The latter interaction, which prevented cleavage-site engagement, also involved base flipping and might represent the sequence-interrogation phase that precedes specific recognition. MspJI is unusual in that DNA molecules are recognized and cleaved by different subunits. Such interchange of function might explain how other complex multimeric restriction enzymes act.     

Direct visualization of the sites of DNA methylation in human,and mosquito chromosomes

Human and mosquito fixed chromosomes were digested with restriction endonucleases that are inhibited by the presence of 5-methylcytosine in their restriction sites (Hha I, Hin PI, Hpa II), and with endonucleases for which cleavage is less dependent on the state of methylation (Taq I, Msp I). Methylation-dependent enzymes extracted low DNA amounts from human chromosomes, while methylation-independent enzymes extracted moderate to high amounts of DNA. After DNA demethylation with 5-azacytidine the isoschizomers Hpa II (methylation-dependent) and Msp I (methylation-independent) extracted 12-fold and 1.4-fold amounts of DNA from human chromosomes, respectively. These findings indicate that human DNA has a high concentration of Hpa II and Msp I restriction sites (CCGG), and that the internal C of this sequence is methylated in most cases, while the external cytosine is methylated less often. All the enzymes tested released moderate amounts of DNA from mosquito chromosomes whether or not the DNA was demethylated with 5-azacytidine. Hpa II induced banding in the centromere chromosome regions. After demethylation with 5-azacytidine this banding disappeared. Mosquito DNA has therefore, moderate to high frequencies of nonmethylated CpG duplets. The only exception is the centromeric DNA, in which the high levels of C methylation present produce cleavage by Hpa II and the appearance of banding. Centromere regions of human chromosomes 1 have a moderately low concentration of Hpa II-Msp I restriction sites.     

Detection of heavy methylation in human repetitive DNA subsets by a monoclonal antibody against 5-methylcytosine

A monoclonal antibody (IgM) against 5-methylcytosine (mC) was isolated and characterized. It showed a high specificity for mC with a cross-reactivity of less than 1% with cytosine and 0.1% with thymidine. An improved immunohybridization method, originally developed with polyclonal antibodies (Sano et al. (1980) Proc. Natl. Acad. Sci. USA 77, 3581), was applied to detect mC in immobilized DNA using the new monoclonal preparation. Human genomic DNA was cleaved with the restriction enzyme EcoRI and successively fractionated by malachite-green affinity chromatography and agarose gel electrophoresis. The fractionated DNA was transferred to nitrocellulose paper and treated with the anti-mC monoclonal antibody. Heavy methylation was observed in EcoRI-ladders of repetitive sequences of 1360, 1750, 2200 and 3400 bp, while 340, 660 and 2700 bp fragments were less methylated. The results show that methylation occurs in limited subsets of satellite II and III repetitive DNAs that contain high amounts of methylatable CpG dinucleotides, or CpG clusters.     

Tetrameric restriction enzymes: expansion to the GIY-YIG nuclease family

The GIY-YIG nuclease domain was originally identified in homing endonucleases and enzymes involved in DNA repair and recombination. Many of the GIY-YIG family enzymes are functional as monomers. We show here that the Cfr42I restriction endonuclease which belongs to the GIY-YIG family and recognizes the symmetric sequence 5′-CCGC/GG-3′ (‘/’ indicates the cleavage site) is a tetramer in solution. Moreover, biochemical and kinetic studies provided here demonstrate that the Cfr42I tetramer is catalytically active only upon simultaneous binding of two copies of its recognition sequence. In that respect Cfr42I resembles the homotetrameric Type IIF restriction enzymes that belong to the distinct PD-(E/D)XK nuclease superfamily. Unlike the PD-(E/D)XK enzymes, the GIY-YIG nuclease Cfr42I accommodates an extremely wide selection of metal-ion cofactors, including Mg²⁺, Mn²⁺, Co²⁺, Zn²⁺, Ni²⁺, Cu²⁺ and Ca²⁺. To our knowledge, Cfr42I is the first tetrameric GIY-YIG family enzyme. Similar structural arrangement and phenotypes displayed by restriction enzymes of the PD-(E/D)XK and GIY-YIG nuclease families point to the functional significance of tetramerization.     

Ssh AI restriction endonuclease from Salmonella shikmonah

A new type II restriction endonuclease, SshAI, was purified from Salmonella shikmonah TK139 of kangaroo origin. The recognition and cleavage specificity of Ssh AI was determined to be 5'-CC/TNAGG-3', identical to that of SauI from Streptomyces aureofaciens and Bsu36I from Bacillus subtilis. Based on closely related and in part overlapping recognition specificities of Ssh AI and other restriction endonucleases, a close evolutionary relationship is proposed for all known Salmonella restriction endonucleases.     

Cloning and expression of the Apa LI, Nsp I, Nsp HI, Sac I, Sca I, and Sap I restriction-modification systems in Escherichia coli

The genes encoding the ApaLI (5′-G^TGCAC-3′), NspI (5′-RCATG^Y-3′), NspHI (5′-RCATG^Y-3′), SacI (5′-GAGCT^C-3′), SapI (5′-GCTCTTCN1^-3′, 5′-^N4GAAGAGC-3′) and ScaI (5′-AGT^ACT-3′) restriction-modification systems have been cloned in E.?coli. Amino acid sequence comparison of M.ApaLI, M.NspI, M.NspHI, and M.SacI with known methylases indicated that they contain the ten conserved motifs characteristic of C5 cytosine methylases. NspI and NspHI restriction-modification systems are highly homologous in amino acid sequence. The C-termini of the NspI and NlaIII (5′-CATG-3′) restriction endonucleases share significant similarity. 5mC modification of the internal C in a SacI site renders it resistant to SacI digestion. External 5mC modification of a SacI site has no effect on SacI digestion. N4mC modification of the second base in the sequence 5′-GCTCTTC-3′ blocks SapI digestion. N4mC modification of the other cytosines in the SapI site does not affect SapI digestion. N4mC modification of ScaI site blocks ScaI digetion. A DNA invertase homolog was found adjacent to the ApaLI restriction-modification system. A DNA transposase subunit homolog was found upstream of the SapI restriction endonuclease gene.     

Specificity of restriction endonucleases and DNA modification methyltransferases a review (Edition 3)

The properties and sources of all known class-I, class-II and class-III restriction endonucleases (ENases) and DNA modification methyltransferases (MTases) are listed and newly subclassified according to their sequence specificity. In addition, the enzymes are distinguished in a novel manner according to sequence specificity, cleavage position and methylation sensitivity. Furthermore, new nomenclature rules are proposed for unambiguously defined enzyme names. In the various Tables, the enzymes are cross-indexed alphabetically according to their names (Table I), classified according to their recognition sequence homologies (Table II), and characterized within Table II by the cleavage and methylation positions, the number of recognition sites on the DNA of the bacteriophages lambda, phi X174, and M13mp7, the viruses Ad2 and SV40, the plasmids pBR322 and pBR328, and the microorganisms from which they originate. Other tabulated properties of the ENases include relaxed specificities (integrated within Table II), the structure of the generated fragment ends (Table III), interconversion of restriction sites (Table IV) and the sensitivity to different kinds of DNA methylation (Table V). Table VI shows the influence of class-II MTases on the activity of class-II ENases with at least partially overlapping recognition sequences. Table VII lists all class-II restriction endonucleases and MTases which are commercially available. The information given in Table V focuses on the influence of methylation of the recognition sequences on the activity of ENases. This information might be useful for the design of cloning experiments especially in Escherichia coli containing M.EcodamI and M.EcodcmI [H16, M21, U3] or for studying the level and distribution of site-specific methylation in cellular DNA, e.g., 5'- (M)CpG-3' in mammals, 5'-(M)CpNpG-3' in plants or 5'-GpA(M)pTpC-3' in enterobacteria [B29, E4, M30, V4, V13, W24]. In Table IV a cross index for the interconversion of two- and four-nt 5'-protruding ends into new recognition sequences is complied. This was obtained by the fill-in reaction with the Klenow (large) fragment of the E. coli DNA polymerase I (PolIk), or additional nuclease S1 treatment followed by ligation of the modified fragment termini [P3]. Interconversion of restriction sites generates novel cloning sites without the need of linkers. This should improve the flexibility of genetic engineering experiments [K56, P3].(ABSTRACT TRUNCATED AT 400 WORDS)     

Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing

Restriction-modification (R-M) systems pose a major barrier to DNA transformation and genetic engineering of bacterial species. Systematic identification of DNA methylation in R-M systems, including N⁶-methyladenine (6mA), 5-methylcytosine (5mC) and N⁴-methylcytosine (4mC), will enable strategies to make these species genetically tractable. Although single-molecule, real time (SMRT) sequencing technology is capable of detecting 4mC directly for any bacterial species regardless of whether an assembled genome exists or not, it is not as scalable to profiling hundreds to thousands of samples compared with the commonly used next-generation sequencing technologies. Here, we present 4mC-Tet-assisted bisulfite-sequencing (4mC-TAB-seq), a next-generation sequencing method that rapidly and cost efficiently reveals the genome-wide locations of 4mC for bacterial species with an available assembled reference genome. In 4mC-TAB-seq, both cytosines and 5mCs are read out as thymines, whereas only 4mCs are read out as cytosines, revealing their specific positions throughout the genome. We applied 4mC-TAB-seq to study the methylation of a member of the hyperthermophilc genus, Caldicellulosiruptor, in which 4mC-related restriction is a major barrier to DNA transformation from other species. In combination with MethylC-seq, both 4mC- and 5mC-containing motifs are identified which can assist in rapid and efficient genetic engineering of these bacteria in the future.     

Isolation of a deoxycytidylate methyl transferase capable of protecting DNA uniquely against cleavage by endonuclease R.Aqu I (isoschizomer of Ava I).

A sequence-specific modification methylase (M.AquI) was isolated and purified from Agmenellum quadruplicatum (Synechococcus PCC 7002). This enzyme uniquely methylates the deoxycytidylate residue in the sequence *CYCGRG indicated by the asterisk. It was shown to protect DNA against cleavage by restriction endonucleases AvaI, SmaI and XhoI, which recognize the sequences CYCGRG, CCCGGG, and CTCGAG, respectively.     

Distribution of 5-methylcytosine residues in 5S rRNA genes in Arabidopsis thaliana and Secale cereale

Bisulfite genomic sequencing was used to localise 5-methylcytosine residues (mC) in 5S rRNA genes of Arabidopsis thaliana and Secale cereale. The maps of mC distribution were compared with the previously published map of the corresponding region in Nicotiana tabacum. In all three species, the level of methylation of 5S rRNA genes was generally higher than the average for the entire genome. The ratio of 5S rDNA methylation to average overall methylation was 44%/30-33% for N. tabacum, 27%/4-6% for A. thaliana and 24%/20-22% for S. cereale. With the exception of one clone from S. cereale, no methylation-free 5S rDNA was detected. The level of methylation at different sequence motifs in 5S rDNA was calculated for N. tabacum/A. thaliana/ S. cereale, and this analysis yielded the following values (expressed as a percentage of total C): mCG 90%/78%/85%, mCWG 89%/41%/53%, mCmCG 72%/32%/16%, mCCG 4%/2%/0%, mCHH 15%/6%/1%, where W=A or T, and H=A or C or T. Non-symmetrical methylation was almost negligible in the large genome of S. cereale but relatively frequent in N. tabacum and A. thaliana, suggesting that the strict correlation between genome size and cytosine methylation might be violated for this type of methylation. Among non-symmetrical motifs the mCWA triplets were significantly over-represented in Arabidopsis, while in tobacco this preference was not as pronounced. The differences in methylation levels in different sequence contexts might be of phylogenetic significance, but further species in related and different taxa need to be studied before firm conclusions can be drawn.     

Engineering of restriction endonucleases: using methylation activity of the bifunctional endonuclease Eco57I to select the mutant with a novel sequence specificity

Type II restriction endonucleases (REs) are widely used tools in molecular biology, biotechnology and diagnostics. Efforts to generate new specificities by structure-guided design and random mutagenesis have been unsuccessful so far. We have developed a new procedure called the methylation activity-based selection (MABS) for generating REs with a new specificity. MABS uses a unique property of bifunctional type II REs to methylate DNA targets they recognize. The procedure includes three steps: (1) conversion of a bifunctional RE into a monofunctional DNA-modifying enzyme by cleavage center disruption; (2) mutagenesis and selection of mutants with altered DNA modification specificity based on their ability to protect predetermined DNA targets; (3) reconstitution of the cleavage center's wild-type structure. The efficiency of the MABS technique was demonstrated by altering the sequence specificity of the bifunctional RE Eco57I from 5'-CTGAAG to 5'-CTGRAG, and thus generating the mutant restriction endonuclease (and DNA methyltransferase) of a specificity not known before. This study provides evidence that MABS is a promising technique for generation of REs with new specificities.