Type IIS restriction endonuclease Eco31I is a ‘short-distance cutter’, which cleaves DNA strands close to its recognition sequence, 5′-GGTCTC(1/5). Previously, it has been proposed that related endonucleases recognizing a common sequence core GTCTC possess two active sites for cleavage of both strands in the DNA substrate. Here, we present bioinformatic identification and experimental evidence for a single nuclease active site. We identified a short region of homology between Eco31I and HNH nucleases, constructed a three-dimensional model of the putative catalytic domain and validated our predictions by random and site-specific mutagenesis. The restriction mechanism of Eco31I is suggested by analogy to the mechanisms of phage T4 endonuclease VII and homing endonuclease I-PpoI. We propose that residues D311 and N334 coordinate the cofactor. H312 acts as a general base activating water molecule for the nucleophilic attack. K337 together with R340 and D345 are located in close proximity to the active center and are essential for correct folding of catalytic motif, while D345 together with R264 and D273 could be directly involved in DNA binding. We also predict that the Eco31I catalytic domain contains a putative Zn-binding site, which is essential for its structural integrity. Our results suggest that the HNH-like active site is involved in the cleavage of both strands in the DNA substrate. On the other hand, analysis of site-specific mutants in the region, previously suggested to harbor the second active site, revealed its irrelevance to the nuclease activity. Thus, our data argue against the earlier prediction and indicate the presence of a single conserved active site in Type IIS restriction endonucleases that recognize common sequence core GTCTC.
restriction endonuclease; Type IIS; HNH; endonuclease VII; active site
A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes and gene products. It provides explicit categories for the many different Type II enzymes now identified and provides a system for naming the putative genes found by sequence analysis of microbial genomes.
Esp1396I restriction–modification (RM) system recognizes an interrupted palindromic DNA sequ ence 5′-CCA(N)5TGG-3′. The Esp1396I RM system was found to reside on pEsp1396, a 5.6 kb plasmid naturally occurring in Enterobacter sp. strain RFL1396. The nucleotide sequence of the entire 5622 bp pEsp1396 plasmid was determined on both strands. Identified genes for DNA methyltransferase (esp1396IM) and restriction endonuclease (esp1396IR) are transcribed convergently. The restriction endonuclease gene is preceded by the small ORF (esp1396IC) that possesses a strong helix-turn-helix motif and resembles regulatory proteins found in PvuII, BamHI and few other RM systems. Gene regulation studies revealed that C.Esp1396I acts as both a repressor of methylase expression and an activator of regulatory protein and restriction endonuclease expression. Our data indicate that C protein from Esp1396I RM system activates the expression of the Enase gene, which is co-transcribed from the promoter of regulatory gene, by the mechanism of coupled translation.
A new type II restriction endonuclease AarI has been isolated from Arthrobacter aurescens SS2-322. AarI recognizes the non-palindromic heptanucleotide sequence 5′-CACCTGC(N)4/8-3′ and makes a staggered cut at the fourth and eighth bases downstream of the target duplex producing a four base 5′-protruding end. AarI activity is stimulated by oligodeoxyribonucleotide duplexes containing an enzyme-specific recognition sequence.
A new type II restriction endonuclease designated PfoI has been partially purified from Pseudomonas fluorescens biovar 126. PfoI recognises the interrupted hexanucleotide palindromic sequence 5′-T↓CCNGGA-3′ and cleaves DNA to produce protruding pentanucleotide 5′-ends.
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 β 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.
A new type II restriction endonuclease designated FspAI has been partially purified from a Flexibacter species Tv-m21K. FspAI recognizes the octanucleotide sequence 5′-RTGC↓GCAY-3′ and cleaves it in the center generating blunt-ended DNA fragments.
A new type II restriction endonuclease designated OliI
has been partially purified from the halophilic bacterium Oceanospirillum
linum 4-5D. OliI recognizes the interrupted
hexanucleotide palindrome 5′-CACNN↓NNGTG-3′ and
cleaves it in the center generating blunt-ended DNA fragments.
We report the properties of the new BseMII restriction and
modification enzymes from Bacillus stearothermophilus Isl
15-111, which recognize the 5′-CTCAG sequence,
and the nucleotide sequence of the genes encoding them. The restriction
endonuclease R.BseMII makes a staggered cut at
the tenth base pair downstream of the recognition sequence on the upper
strand, producing a two base 3′-protruding end.
Magnesium ions and S-adenosyl-l-methionine (AdoMet)
are required for cleavage. S-adenosylhomocysteine
and sinefungin can replace AdoMet in the cleavage reaction. The BseMII methyltransferase modifies unique adenine
residues in both strands of the target sequence 5′-CTCAG-3′/5′-CTGAG-3′. Monomeric R.BseMII
in addition to endonucleolytic activity also possesses methyltransferase
activity that modifies the A base only within the 5′-CTCAG
strand of the target duplex. The deduced amino acid sequence of the
restriction endonuclease contains conserved motifs of DNA N6-adenine
methylases involved in S-adenosyl-l-methionine
binding and catalysis. According to its structure and enzymatic
properties, R.BseMII may be regarded as a representative
of the type IV restriction endonucleases.
Methyltransferases (MTases) of procaryotes affect general cellular processes such as mismatch repair, regulation of transcription, replication, and transposition, and in some cases may be essential for viability. As components of restriction-modification systems, they contribute to bacterial genetic diversity. The genome of Helicobacter pylori strain 26695 contains 25 open reading frames encoding putative DNA MTases. To assess which MTase genes are active, strain 26695 genomic DNA was tested for cleavage by 147 restriction endonucleases; 24 were found that did not cleave this DNA. The specificities of 11 expressed MTases and the genes encoding them were identified from this restriction data, combined with the known sensitivities of restriction endonucleases to specific DNA modification, homology searches, gene cloning and genomic mapping of the methylated bases m4C, m5C, and m6A.
Kpn 2I enzymes of a type II restriction-modification (R-M) system from the bacterium Klebsiella pneumoniae strain RFL2 recognize the sequence 5'-TCCGGA-3'. The Kpn 2I R-M genes have been cloned and expressed in Escherichia coli. DNA sequence analysis revealed the presence of two convergently transcribed open reading frames (ORFs) coding for a restriction endonuclease (Enase) of 301 amino acids (34. 8 kDa) and methyltransferase (Mtase) of 375 amino acids (42.1 kDa). The 3'-terminal ends of these genes ( kpn2IR and kpn2IM, respectively) overlap by 11 bp. In addition, a small ORF (gene kpn2IC ) capable of coding for a protein of 96 amino acids in length (10.6 kDa) was found upstream of kpn2IM. The direction of kpn2IC transcription is opposite to that of kpn2IM. The predicted amino acid sequence of this ORF includes a probable helix-turn-helix motif. We show that the product of kpn2IC represses expression of the Kpn 2I Mtase but has no influence on expression of the Enase gene. Such a mode of regulation is unique among R-M systems analyzed so far. The Kpn 2I R-M is located on the K.pneumoniae RFL2 plasmid pKp4.3, which is able to replicate in E.coli cells.
A new restriction endonuclease Bse SI has been isolated from Bacillus stearothermophilus Jo10-553. Bse SI recognizes a degenerate hexanucleotide sequence 5'-G(G/T)GC(A/C)C-3' and cleaves DNA to produce 3[prime]-protruding tetranucleotide ends.
A new restriction endonuclease Abe I has beenisolated from Azotobacter beijerinckii. This enzymerecognizes the asymmetric heptanucleotide sequence 5'-CCTCAGC-3' and cleaves within it symmetrically at positions -5/-2 in the opposing strands, producing three base protruding 5'-ends.
A new type IIS restriction endonuclease Bfi I hasbeen partially purified from Bacillus firmus S8120. Bfi I recognizes the non-palindromic hexanucleotide sequence 5'-ACTGGG(N)5/4-3' and makes a staggered cut at the fifth base pair downstream of the recognition sequence on the upper strand, producing a single base 3' protruding end.
The Bpu 10I R-M system from Bacillus pumilus 10, which recognizes the asymmetric 5'-CCTNAGC sequence, has been cloned, sequenced and expressed in Escherichia coli . The system comprises four adjacent, similarly oriented genes encoding two m5C MTases and two subunits of Bpu 10I ENase (34.5 and 34 kDa). Both bpu10IR genes either in cis or trans are needed for the manifestation of R. Bpu 10I activity. Subunits of R. Bpu 10I, purified to apparent homogeneity, are both required for cleavage activity. This heterosubunit structure distinguishes the Bpu 10I restriction endonuclease from all other type II restriction enzymes described previously. The subunits reveal 25% amino acid identity. Significant similarity was also identified between a 43 amino acid region of R. Dde I and one of the regions of higher identity shared between the Bpu 10I subunits, a region that could possibly include the catalytic/Mg2+binding center. The similarity between Bpu 10I and Dde I MTases is not limited to the conserved motifs (CM) typical for m5C MTases. It extends into the variable region that lies between CMs VIII and IX. Duplication of a progenitor gene, encoding an enzyme recognizing a symmetric nucleotide sequence, followed by concerted divergent evolution, may provide a possible scenario leading to the emergence of the Bpu 10I ENase, which recognizes an overall asymmetric sequence and cleaves within it symmetrically.
Bcg I and Bcg I-like restriction endonucleases cleave double stranded DNA specifically on both sides of their asymmetric recognition sequences which are interrupted by several ambiguous base pairs. Their heterosubunit structure, bifunctionality and stimulation by AdoMet make them different from other classified restriction enzymes. Here we report on a new Bcg I-like restriction endonuclease, Bpl I from Bacillus pumilus , which in contrast to all other Bcg I-like enzymes, recognizes a symmetric interrupted sequence, and which, like Bcg I, cleaves double stranded DNA upstream and downstream of its recognition sequence (8/13)GAGN5CTC(13/8). Like Bcg I, Bpl I is a bifunctional enzyme revealing both DNA cleavage and methyltransferase activities. There are two polypeptides in the homogeneous preparation of Bpl I with molecular masses of approximately 74 and 37 kDa. The sizes of the Bpl I subunits are close to those of Bcg I, but the proportion 1:1 in the final preparation is different from that of 2:1 in Bcg I. Low activity observed with Mg2+increases >100-fold in the presence of AdoMet. Even with AdoMet though, specific cleavage is incomplete. S -adenosylhomocysteine (AdoHcy) or sinefungin can replace AdoMet in the cleavage reaction. AdoHcy activated Bpl I yields complete cleavage of DNA.
The genomic region encoding the type IIS restriction-modification (R-M) system HphI (enzymes recognizing the asymmetric sequence 5'-GGTGA-3'/5'-TCACC-3') from Haemophilus parahaemolyticus were cloned into Escherichia coli and sequenced. Sequence analysis of the R-M HphI system revealed three adjacent genes aligned in the same orientation: a cytosine 5 methyltransferase (gene hphIMC), an adenine N6 methyltransferase (hphIMA) and the HphI restriction endonuclease (gene hphIR). Either methyltransferase is capable of protecting plasmid DNA in vivo against the action of the cognate restriction endonuclease. hphIMA methylation renders plasmid DNA resistant to R.Hindill at overlapping sites, suggesting that the adenine methyltransferase modifies the 3'-terminal A residue on the GGTGA strand. Strong homology was found between the N-terminal part of the m6A methyltransferasease and an unidentified reading frame interrupted by an incomplete gaIE gene of Neisseria meningitidis. The HphI R-M genes are flanked by a copy of a 56 bp direct nucleotide repeat on each side. Similar sequences have also been identified in the non-coding regions of H.influenzae Rd DNA. Possible involvement of the repeat sequences in the mobility of the HphI R-M system is discussed.
CfrI, a new restriction endonuclease of unique substrate specificity, has been isolated from a Citrobacter freundii strain. The enzyme recognizes a degenerated sequence PyGGCCPu in double-strand DNA and cleaves it between Py and G residues to yield 5′ -protruding tetranucleotide ends GGCC.
The specificity of three DNA methyltransferases M.Alw26I, M.Eco31I and M.Esp3I, isolated from Acinetobacter Iwoffi RFL26, Escherichia coli RFL31 and Hafnia alvei RFL3+, respectively, was determined. All the enzymes methylate both strands of asymmetric recognition sites yielding m5C in the top-strand and m6A in the bottom-strand, as below: 5'-GTm5CTC 5'-GGTm5CTC 5'-CGTm5CTC 3'-Cm6AGAG 3'-CCm6AGAG 3'-GCm6AGAG (M.Alw26I) (M.Eco31I) (M.Esp3I) They are the first members of type IIs methyltransferases that modify different types of nucleotides in the recognition sequence.
The cleavage specificity of R.Cfr9I was determined to be C decreases CCGGG whereas the methylation specificity of M.Cfr9I was C4mCCGGG. The action of MspI, HpaII, SmaI, XmaI and Cfr9I restriction endonucleases on an unmethylated parent d(GGACCCGGGTCC) dodecanucleotide duplex and a set of oligonucleotide duplexes, containing all possible substitutions of either 4mC or 5mC for C in the CCCGGG sequence, was investigated. It was found that 4mC methylation, in contrast to 5mC, renders the CCCGGG site resistant to practically all the investigated endonucleases. The cleavage of methylated substrates with restriction endonucleases is discussed.
The Eco57I restriction endonuclease and methylase were purified to homogeneity from the E.coli RR1 strain carrying the eco57IRM genes on a recombinant plasmid. The molecular weight of the denaturated methylase is 63 kDa. The restriction endonuclease exists in a monomeric form with an apparent molecular weight of 104-108 kDa. R.Eco57I also possesses methylase activity. The methylation activities of both enzymes modify the outer A residue in the target sequence 5'CTGAAG yielding N6-methyladenine. M.Eco57I modifies both strands of the substrate while R.Eco57I modifies only one. Only the methylase enzyme is stimulated by Ca2+. The restriction endonuclease shows an absolute requirement for Mg2+ and is stimulated by AdoMet. ATP has no influence on either activity of the enzymes. The subunit structure and enzymatic properties of the Eco57I enzymes distinguish them from all other restriction-modification enzymes that have been described previously. Therefore, RM.Eco57I may be regarded as a representative of a novel class of restriction-modification systems, and we propose to classify it as type IV.
The synthesis of N4-methyl-2'-deoxycytidine and its fully protected mononucleotide, suitable for the oligonucleotide synthesis by phosphotriester method is described. A set of octanucleotides - d(CGCGCGCG), d(CG5mCGCGCG), d(CG4mCGCGCG) and dodecanucleotides - d(GGACCCGGGTCC), d(GGA5mCCCGGGTCC), d(GGA4mCCCGGGTCC) has been synthesized in a solution. Physical characterization of the oligonucleotide duplexes by means of UV and CD spectrometry provides the evidence that 4mC similarly to 5mC favours the B--greater than Z transition, although both of these methylated cytosines inhibit the B--greater than A conformational change. N4-Methylcytosine in contrast to 5-methylcytosine reduces the DNA double helix thermal stability.
The sequences coding for DNA[cytosine-N4]methyltransferases MvaI (from Micrococcus varians RFL19) and Cfr9I (from Citrobacter freundii RFL9) have been determined. The predicted methylases are proteins of 454 and 300 amino acids, respectively. Primary structure comparison of M.Cfr9I and another m4C-forming methylase, M.Pvu II, revealed extended regions of homology. The sequence comparison of the three DNA[cytosine-N4]-methylases using originally developed software revealed two conserved patterns, DPF-GSGT and TSPPY, which were found similar also to those of adenine and DNA[cytosine-C5]-methylases. These data provided a basis for global alignment and classification of DNA-methylase sequences. Structural considerations led us to suggest that the first region could be the binding site of AdoMet, while the second is thought to be directly involved in the modification of the exocyclic amino group.