Evolutionary relationship of Alw26I, Eco31I and Esp3I, restriction endonucleases that recognise overlapping sequences.

Type II restriction endonucleases (ENases) have served as models for understanding the enzyme-based site-specific cleavage of DNA. Using the knowledge gained from the available crystal structures, a number of attempts have been made to alter the specificity of ENases by mutagenesis. The negative results of these experiments argue that the three-dimensional structure of DNA-ENase complexes does not provide enough information to enable us to understand the interactions between DNA and ENases in detail. This conclusion calls for alternative approaches to the study of structure-function relationships related to the specificity of ENases. Comparative analysis of ENases that manifest divergent substrate specificities, but at the same time are evolutionarily related to each other, may be helpful in this respect. The success of such studies depends to a great extent on the availability of related ENases that recognise partially overlapping nucleotide sequences (e.g. sets of enzymes that bind to recognition sites of increasing length). In this study we report the cloning and sequence analysis of genes for three Type IIS restriction-modification (RM) systems. The genes encoding the ENases Alw26I, Eco31I and Esp3I (whose recognition sequences are 5'-GTCTC-3', 5'-GGTCTC-3' and 5'-CGTCTC-3', respectively) and their accompanying methyltransferases (MTases) have been cloned and the deduced amino acid sequences of their products have been compared. In pairwise comparisons, the degree of sequence identity between Alw26I, Eco31I and Esp3I ENases is higher than that observed hitherto among ENases that recognise partially overlapping nucleotide sequences. The sequences of Alw26I, Eco31I and Esp3I also reveal identical mosaic patterns of sequence conservation, which supports the idea that they are evolutionarily related and suggests that they should show a high level of structural similarity. Thus these ENases represent very attractive models for the study of the molecular basis of variation in the specific recognition of DNA targets. The corresponding MTases are represented by proteins of unusual structural and functional organisation. Both M. Alw26I and M. Esp3I are represented by a single bifunctional protein, which is composed of an m(6)A-MTase domain fused to a m(5)C-MTase domain. In contrast, two separate genes encode the m(6)A-MTase and m(5)C-MTase in the Eco31I RM system. Among the known bacterial m(5)C-MTases, the m(5)C-MTases of M. Alw26I, M. Eco31I and M. Esp3I represent unique examples of the circular permutation of their putative target recognition domains together with the conserved motifs IX and X.

Characterization of AloI, a restriction-modification system of a new type.

We report the properties of the new AloI restriction and modification enzyme from Acinetobacter lwoffi Ks 4-8 that recognizes the DNA target 5' GGA(N)6GTTC3' (complementary strand 5' GAAC(N)6TCC3'), and the nucleotide sequence of the gene encoding this enzyme. AloI is a bifunctional large polypeptide (deduced M(r) 143 kDa) revealing both DNA endonuclease and methyltransferase activities. Depending on reaction cofactors, AloI cleaves double-stranded DNA on both strands, seven bases on the 5' side, and 12-13 bases on the 3' side of its recognition sequence, and modifies adenine residues in both DNA strands in the target sequence yielding N6-methyladenine. For cleavage activity AloI maintains an absolute requirement for Mg(2+) and does not depend on or is stimulated by either ATP or S-adenosyl-L-methionine. Modification function requires the presence of S-adenosyl-L-methionine and is stimulated by metal ions (Ca(2+)). The C-terminal and central parts of the protein were found to be homologous to certain specificity (HsdS) and modification (HsdM) subunits of type I R-M systems, respectively. The N-terminal part of the protein possesses the putative endonucleolytic motif DXnEXK of restriction endonucleases. The deduced amino acid sequence of AloI shares significant homology with polypeptides encoding HaeIV and CjeI restriction-modification proteins at the N-terminal and central, but not at the C-terminal domains. The organization of AloI implies that its evolution involved fusion of an endonuclease and the two subunits, HsdM and HsdS, of type I restriction enzymes. According to the structure and function properties AloI may be regarded as one more representative of a newly emerging group of HaeIV-like restriction endonucleases. Discovery of these enzymes opens new opportunities for constructing restriction endonucleases with a new specificity.

Characterization of BseMII, a new type IV restriction-modification system, which recognizes the pentanucleotide sequence 5'-CTCAG(N)(10/8)/.

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.

Novel subtype of type IIs restriction enzymes. BfiI endonuclease exhibits similarities to the EDTA-resistant nuclease Nuc of Salmonella typhimurium.

The type IIs restriction enzyme BfiI recognizes the non-palindromic nucleotide sequence 5'-ACTGGG-3' and cleaves complementary DNA strands 5/4 nucleotides downstream of the recognition sequence. The genes coding for the BfiI restriction-modification (R-M) system were cloned/sequenced and biochemical characterization of BfiI restriction enzyme was performed. The BfiI R-M system contained three proteins: two N4-methylcytosine methyltransferases and a restriction enzyme. Sequencing of bisulfite-treated methylated DNA indicated that each methyltransferase modifies cytosines on opposite strands of the recognition sequence. The N-terminal part of the BfiI restriction enzyme amino acid sequence revealed intriguing similarities to an EDTA-resistant nuclease of Salmonella typhimurium. Biochemical analyses demonstrated that BfiI, like the nuclease of S. typhimurium, cleaves DNA in the absence of Mg(2+) ions and hydrolyzes an artificial substrate bis(p-nitrophenyl) phosphate. However, unlike the nonspecific S. typhimurium nuclease, BfiI restriction enzyme cleaves DNA specifically. We propose that the DNA-binding specificity of BfiI stems from the C-terminal part of the protein. The catalytic N-terminal subdomain of BfiI radically differs from that of type II restriction enzymes and is presumably similar to the EDTA-resistant nonspecific nuclease of S. typhimurium; therefore, BfiI did not require metal ions for catalysis. We suggest that BfiI represents a novel subclass of type IIs restriction enzymes that differs from the archetypal FokI endonuclease by the fold of its cleavage domain, the domain location, and reaction mechanism.

Structural organization and regulation of the plasmid-borne type II restriction-modification system Kpn2I from Klebsiella pneumoniae RFL2.

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.

Cloning and analysis of the four genes coding for Bpu10I restriction-modification enzymes.

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.

Cloning and analysis of the genes encoding the type IIS restriction-modification system HphI from Haemophilus parahaemolyticus.

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.

DNA restriction-modification systems mediate plasmid maintenance.

Two plasmid-carried restriction-modification (R-M) systems, EcoRI (from pMB1 of Escherichia coli) and Bsp6I (from pXH13 of Bacillus sp. strain RFL6), enhance plasmid segregational stability in E. coli and Bacillus subtilis, respectively. Inactivation of the endonuclease or the presence of the methylase in trans abolish the stabilizing activity of the R-M systems. We propose that R-M systems mediate plasmid segregational stability by postsegregational killing of plasmid-free cells. Plasmid-encoded methyltransferase modifies host DNA and thus prevents its digestion by the restriction endonuclease. Plasmid loss entails degradation and/or dilution of the methylase during cell growth and appearance of unmethylated sites in the chromosome. Double-strand breaks, introduced at these sites by the endonuclease, eventually cause the death of the plasmid-free cells. Contribution to plasmid stability is a previously unrecognized biological role of the R-M systems.

Cloning and analysis of the plasmid-borne genes encoding the Bsp6I restriction and modification enzymes.

The Bsp6I restriction and modification (R-M) system has been localized on the plasmid pXH13, naturally occurring in the Bacillus sp. strain RFL6. The genes coding for the Bsp6I R-M system, a Fnu4HI isoschizomer recognizing the sequence GCNGC, have been cloned in Escherichia coli by two steps. The nucleotide sequence of a 2126-bp region containing the genes for restriction endonuclease (ENase; bsp6IR) and DNA methyltransferase (MTase; bsp6IM) has been determined. The genes are separated by 99 bp and are arranged tandemly with bsp6IR preceding bsp6IM. The DNA sequence predicts an ENase of 174 amino acids (aa) (19.9 kDa) and a MTase of 315 aa (36.3 kDa). M.Bsp6I contains all the conserved aa sequence motifs characteristic for m5C-MTases. In addition, its variable region exhibits a slight similarity to the 5'-GCNGC-3'-specific target-recognition domain (TRD) from M.phi 3T. No aa sequence similarity was found between R.Bsp6I and M.Bsp6I, nor among R.Bsp6I and other known ENases. We have tested recombinant plasmids carrying the complete R-M system for their ability to transform native and pre-methylated Escherichia coli hosts. The results indicate that pre-methylation increases the efficiency of establishment of the complete R-M system. In addition, we have obtained orientation-dependent differences in transformation efficiency.

Cloning and characterization of the unusual restriction-modification system comprising two restriction endonucleases and one methyltransferase.

An Escherichia coli RFL47 DNA fragment containing the Eco47IR and Eco47II restriction-modification (R-M) system has been cloned and sequenced. A clone carrying this system has been selected by its ability to restrict phage lambda in vivo. The sequence of 5360 bp was determined, and its analysis revealed three major open reading frames (ORF) corresponding to two restriction endonucleases (ENases) and one DNA methyltransferase (MTase): R.Eco47II (239 amino acid (aa)), R.Eco47I (230 aa) and M.Eco47II (417 aa). The M.Eco47II aa sequence possesses all conserved domains typical for m5C MTases and its variable region has a high homology with M.Sau96I and M.SinI. The ORF harboring a predicted helix-turn-helix motif upstream from the eco47IR gene has been found. No sequence resembling the eco47IM gene has been detected in the complete fragment sequenced, although disrupted ORF, possibly corresponding to the transposase-encoding gene, has been found in the intergenic area between eco47IIM and eco47IR. No homology was found between the ENases; however, both revealed homology with their isoschizomers, R.SinI and R.Sau96I.

Organization and sequence of the HpaII restriction-modification system and adjacent genes.

We report the organization of the HpaII restriction and modification (R-M) system from Haemophilus parainfluenzae (recognition sequence: 5'...CCGG...3'), the sequence of the gene coding for the HpaII restriction endonuclease, and the sequence of the upstream flanking DNA. The HpaII system comprises two genes, hpaIIM, coding for the methyltransferase (MTase; 358 amino acids (aa), 40.4 kDa: product, Cm5CGG), and hpaIIR, coding for the restriction endonuclease (ENase; 358 aa, 40.9 kDa: product, C'CGG). The genes are adjacent, they have the same orientation, and they occur in the order hpaIIM then hpaIIR. The ENase bears little as sequence similarity to the isoschizomeric R.BsuFI and R.MspI ENases. Upstream of, and partly overlapping hpaIIM is the coding sequence for a 141-aa protein that resembles the very-short-patch-repair endonuclease (Vsr) of Escherichia coli. Upstream of that is the coding sequence for a protein that resembles valyl-tRNA synthetase (ValS).

Cloning and analysis of translational control for genes encoding the Cfr9I restriction-modification system.

The complete type-II Cfr9I restriction-modification (R-M) system of Citrobacter freundii strain RFL9, recognizing the DNA sequence CCCGGG, has been cloned and expressed, and functionally active enzymes have been produced in Escherichia coli. Both the methyltransferase (MTase; M.Cfr9I) and restriction endonuclease (ENase; R.Cfr9I) were found to be encoded on a 2.3-kb cloned fragment in the same transcriptional orientation, but differing in translational phases. The last codon (underlined) (ATGA) of the MTase-encoding gene (Cfr9IM) overlaps with the start codon for the ENase-encoding gene (overlined) (cfr9IR). A nucleotide sequence complementary to a predicted Shine-Dalgarno sequence preceding cfr9IR is within this gene. Predicted free energy (delta G) for formation of the mRNA secondary structure involving these complementary sequences was found to be -16.1 kcal/mol. Amino-acid sequence homology of 80% was found between R.Cfr9I and R.XcyI.