A homology model of restriction endonuclease SfiI in complex with DNA

Background

Restriction enzymes (REases) are commercial reagents commonly used in recombinant DNA technologies. They are attractive models for studying protein-DNA interactions and valuable targets for protein engineering. They are, however, extremely divergent: the amino acid sequence of a typical REase usually shows no detectable similarities to any other proteins, with rare exceptions of other REases that recognize identical or very similar sequences. From structural analyses and bioinformatics studies it has been learned that some REases belong to at least four unrelated and structurally distinct superfamilies of nucleases, PD-DxK, PLD, HNH, and GIY-YIG. Hence, they are extremely hard targets for structure prediction and homology-based inference of sequence-function relationships and the great majority of REases remain structurally and evolutionarily unclassified.

Results

SfiI is a REase which recognizes the interrupted palindromic sequence 5'GGCCNNNN^NGGCC3' and generates 3 nt long 3' overhangs upon cleavage. SfiI is an archetypal Type IIF enzyme, which functions as a tetramer and cleaves two copies of the recognition site in a concerted manner. Its sequence shows no similarity to other proteins and nothing is known about the localization of its active site or residues important for oligomerization. Using the threading approach for protein fold-recognition, we identified a remote relationship between SfiI and BglI, a dimeric Type IIP restriction enzyme from the PD-DxK superfamily of nucleases, which recognizes the 5'GCCNNNN^NGGC3' sequence and whose structure in complex with the substrate DNA is available. We constructed a homology model of SfiI in complex with its target sequence and used it to predict residues important for dimerization, tetramerization, DNA binding and catalysis.

Conclusions

The bioinformatics analysis suggest that SfiI, a Type IIF enzyme, is more closely related to BglI, an "orthodox" Type IIP restriction enzyme, than to any other REase, including other Type IIF REases with known structures, such as NgoMIV. NgoMIV and BglI belong to two different, very remotely related branches of the PD-DxK superfamily: the α-class (EcoRI-like), and the β-class (EcoRV-like), respectively. Thus, our analysis provides evidence that the ability to tetramerize and cut the two DNA sequences in a concerted manner was developed independently at least two times in the evolution of the PD-DxK superfamily of REases. The model of SfiI will also serve as a convenient platform for further experimental analyses.     

Crystal structure of restriction endonuclease BglI bound to its interrupted DNA recognition sequence.

The crystal structure of the type II restriction endonuclease BglI bound to DNA containing its specific recognition sequence has been determined at 2.2 A resolution. This is the first structure of a restriction endonuclease that recognizes and cleaves an interrupted DNA sequence, producing 3' overhanging ends. BglI is a homodimer that binds its specific DNA sequence with the minor groove facing the protein. Parts of the enzyme reach into both the major and minor grooves to contact the edges of the bases within the recognition half-sites. The arrangement of active site residues is strikingly similar to other restriction endonucleases, but the co-ordination of two calcium ions at the active site gives new insight into the catalytic mechanism. Surprisingly, the core of a BglI subunit displays a striking similarity to subunits of EcoRV and PvuII, but the dimer structure is dramatically different. The BglI-DNA complex demonstrates, for the first time, that a conserved subunit fold can dimerize in more than one way, resulting in different DNA cleavage patterns.     

Recognition sequence for the restriction endonuclease BglI from Bacillus globigii

Restriction endonuclease BglI recognizes the DNA sequence (Formula: see text) and cleaves each strand at the site indicated, thus generating 3' protruding ends. The recognition sequence was deduced by correlating mapping data with nucleotide sequence information and the position of cleavage was unambiguously determined by 32P labeling of 5' termini produced by BglI digestion.     

A model of restriction endonuclease MvaI in complex with DNA: a template for interpretation of experimental data and a guide for specificity engineering

R.MvaI is a Type II restriction enzyme (REase), which specifically recognizes the pentanucleotide DNA sequence 5'-CCWGG-3' (W indicates A or T). It belongs to a family of enzymes, which recognize related sequences, including 5'-CCSGG-3' (S indicates G or C) in the case of R.BcnI, or 5'-CCNGG-3' (where N indicates any nucleoside) in the case of R.ScrFI. REases from this family hydrolyze the phosphodiester bond in the DNA between the 2nd and 3rd base in both strands, thereby generating a double strand break with 5'-protruding single nucleotides. So far, no crystal structures of REases with similar cleavage patterns have been solved. Characterization of sequence-structure-function relationships in this family would facilitate understanding of evolution of sequence specificity among REases and could aid in engineering of enzymes with new specificities. However, sequences of R.MvaI or its homologs show no significant similarity to any proteins with known structures, thus precluding straightforward comparative modeling. We used a fold recognition approach to identify a remote relationship between R.MvaI and the structure of DNA repair enzyme MutH, which belongs to the PD-(D/E)XK superfamily together with many other REases. We constructed a homology model of R.MvaI and used it to predict functionally important amino acid residues and the mode of interaction with the DNA. In particular, we predict that only one active site of R.MvaI interacts with the DNA target at a time, and the cleavage of both strands (5'-CCAGG-3' and 5'-CCTGG-3') is achieved by two independent catalytic events. The model is in good agreement with the available experimental data and will serve as a template for further analyses of R.MvaI, R.BcnI, R.ScrFI and other related enzymes.     

The recognition site of type II restriction enzyme BglI is interrupted

The Type II restriction endonuclease BglI recognizes the interrupted DNA sequence 5'-G-C-C-N-N-N-N-N-G-G-C-. This sequence occurs at all locations in over 33 000 base pairs of DNA sequence where the enzyme was found to cut DNA and nowhere else. All six of the specified bases are essential parts of the site since all groups of five of the six bases occur in the DNA sequences tested and none of them are cut by BglI. The length of the block of intervening unspecified positions must be exactly five since all other sizes between zero and 15 occur in the DNA sequences searched and none are cut by BglI. The 5'-terminal nucleotides of BglI cleaved phage G4 replicative form DNA and plasmid pER18 DNA were compared with the DNA sequences near the BglI sites on these DNAs. These results indicated that BglI cuts within the intervening unspecified region and produces single-stranded 3' termini that are three bases long. The BglI recognition site and cleavage points can thus be represented as follows: (Formula: see text). This study of the BglI recognition site was facilitated by the use of inexpensive microcomputers. A system of programs was developed that allowed analysis of over 33 kb of DNA sequences stored on flexible magnetic disks or audio cassettes. While these programs were generally written in the higher level language BASIC, some assembly language subroutines were utilized to reduce execution time.     

Structural integrity of the beta beta alpha-Metal finger motif is required for DNA binding and stable protein-DNA complex formation in R.KpnI

Restriction endonuclease (REase) R.KpnI from Klebsiella pneumoniae is a homodimeric enzyme, which recognizes palindromic sequence GGTAC|C and cleaves generating 4 base 3' end overhangs. R.KpnI belongs to the HNH superfamily of nucleases, which are characterized by the presence of the beta beta alpha-Me finger motif. Structurally, this motif consists of a twisted beta-hairpin followed by an alpha-helix, and serves as a scaffold for side chains of residues involved in co-ordination of a divalent metal ion that is required for catalysis. Homology modeling studies of R.KpnI suggested a crossover structure for the alpha-helix, which could possibly form dimeric interface and/or structural scaffold for the active site. We have evaluated the role of the residues present in this alpha-helix in intersubunit interactions and/or stabilization of the active site. We show here that mutations of residues in the alpha-helix lead to a loss of the enzyme activity, but not dimerization ability. Intrinsic fluorescence and circular dichroism studies revealed that the loss of function phenotype was due to the structural perturbation of the beta beta alpha-Me finger motif. The results of mutational analysis suggest that the alpha-helix of the beta beta alpha-Me finger of R.KpnI plays an important role for the stability of the protein-DNA complex.     

Identification of a new subfamily of HNH nucleases and experimental characterization of a representative member, HphI restriction endonuclease

The restriction endonuclease (REase) R. HphI is a Type IIS enzyme that recognizes the asymmetric target DNA sequence 5'-GGTGA-3' and in the presence of Mg(2+) hydrolyzes phosphodiester bonds in both strands of the DNA at a distance of 8 nucleotides towards the 3' side of the target, producing a 1 nucleotide 3'-staggered cut in an unspecified sequence at this position. REases are typically ORFans that exhibit little similarity to each other and to any proteins in the database. However, bioinformatics analyses revealed that R.HphI is a member of a relatively big sequence family with a conserved C-terminal domain and a variable N-terminal domain. We predict that the C-terminal domains of proteins from this family correspond to the nuclease domain of the HNH superfamily rather than to the most common PD-(D/E)XK superfamily of nucleases. We constructed a three-dimensional model of the R.HphI catalytic domain and validated our predictions by site-directed mutagenesis and studies of DNA-binding and catalytic activities of the mutant proteins. We also analyzed the genomic neighborhood of R.HphI homologs and found that putative nucleases accompanied by a DNA methyltransferase (i.e. predicted REases) do not form a single group on a phylogenetic tree, but are dispersed among free-standing putative nucleases. This suggests that nucleases from the HNH superfamily were independently recruited to become REases in the context of RM systems multiple times in the evolution and that members of the HNH superfamily may be much more frequent among the so far unassigned REase sequences than previously thought.     

Crystal structure of MunI restriction endonuclease in complex with cognate DNA at 1.7 A resolution.

The MunI restriction enzyme recognizes the palindromic hexanucleotide sequence C/AATTG (the '/' indicates the cleavage site). The crystal structure of its active site mutant D83A bound to cognate DNA has been determined at 1.7 A resolution. Base-specific contacts between MunI and DNA occur exclusively in the major groove. While DNA-binding sites of most other restriction enzymes are comprised of discontinuous sequence segments, MunI combines all residues involved in the base-specific contacts within one short stretch (residues R115-R121) located at the N-terminal region of the 3(10)4 helix. The outer CG base pair of the recognition sequence is recognized solely by R115 through hydrogen bonds made by backbone and side chain atoms to both bases. The mechanism of recognition of the central AATT nucleotides by MunI is similar to that of EcoRI, which recognizes the G/AATTC sequence. The local conformation of AATT deviates from the typical B-DNA form and is remarkably similar to EcoRI-DNA. It appears to be essential for specific hydrogen bonding and recognition by MunI and EcoRI.     

On the nature of the cytosine-methylated sequence in DNA of Bacillus brevis var. G.-B.

On growing the cells of Bacillus brevis S methionine-auxotroph mutant in the presence of [Me-3H]methionine, practically all the radioactivity incorporated into DNA is found to exist in 5-methylcytosine and N6-methyladenine. The analysis of pyrimidine isopliths isolated from DNA shows that radioactivity only exists in mono- and dinucleotides and the content of 5-methylcytosine in R-m5 C-R and R-m5 C-T-R oligonucleotides is equal. The analysis of dinucleotides isolated from DNA by means of pancreatic DNAase hydrolysis allows the nature of purine residues neighbouring 5-methylcytosine to be identified and shows that 5-methylcytosine localizes in G-m5 C-A and G-m5 C-Tr fragments. B. brevis S DNA methylase modifying cytosine residues recognizes the GCA/TGC degenerate nucleotide sequence which is a part of the following complementary structure with a two-fold rotational axis of symmetry: (5')...N'-G-C-T-G-C-N... (3') (3')...N-C-G-A-C-G-N'... (5') (Methylated cytosine residues are askerisked). Cytosine-modifying DNA methylase activity is isolated from B. brevis cells; it is capable of methylating in vitro homologous and heterologous DNA. Hence DNA in bacterial cells can be undermethylated. This enzyme methylates cytosine residues in native and denatured DNA in the same nucleotide sequences. Specificity of methylation of cytosine residues in vitro and in vivo does not depend on the nature of substrate DNA. DNA methylases of different variants of B. brevis (R, S, P+, P-)) methylate cytosine residues in the same nucleotide sequences. It means that specificity or methylation of DNA cytosine residues in the cells of different variants of B. brevis is the same.     

MamI, a novel class-II restriction endonuclease from Microbacterium ammoniaphilum recognizing 5'-GATNN decreases NNATC-3'

A new site-specific class-II restriction endonuclease, MamI, has been discovered in the nonsporulating Gram+ Microbacterium ammoniaphilum. MamI recognition sequence and cleavage positions were deduced using experimental and computer-assisted mapping and sequencing approaches. MamI cleavage specificity corresponds to: [formula: see text] The novel 43-kD enzyme recognizes a palindromic hexanucleotide interrupted by four ambiguous nucleotides. MamI cleavage positions are located in the center of the recognition sequence resulting in blunt-ended fragments after cleavage in the presence of Mg2+ ions. MamI is inhibited by N6-methyladenine residues. In case of overlapping sequences of MamI and Escherichia coli-coded DNA modification methyltransferase M.EcodamI (5'-[formula: see text]-3'), cleavage of DNA isolated from E. coli wild-type cells will be inhibited. By applying incubation conditions forcing star activity, relaxing of MamI sequence specificity is observed (MamI*).     

Specificity of methylation of cytosine risidues in DNA of Bacillus brevis var. G-B]

On growing the cells of Bacillus brevis S methionine-auxotroph mutant in the presence of (methyl-3H)-methionine practically the total radioactivity included into DNA is found to exist in 5-methylcytosine (MC) and 6N-methyladenine (MA). The analysis of pyrimidine isopliths isolated from DNA shows that radioactivity only exists in mono- and dinucleotides and the content of MC in Pur-MC-Pur and Pur-MC-T-Pur oligonucleotides is equal. The analysis of dinucleotides isolated from DNA by means of pancreatic DNAase hydrolysis allows the nature of purine residues neighbouring with MC to be revealed and shows that MC localizes in G-MC-A and G-MC-T-Pu fragments. Bac. brevis S DNA-methylase modifying cytosine residues recognizes the GCAT GC degenerative nucleotide sequence which is a part of the following complementary structure with rotational symmetry: (5') ... N'--G--MC--T--G--C--N ... (3') (3') ... N--C--G--A--MC--G--N' ... (5') Cytosine modifying DNA-methylase activity is isolated from Bac. brevis cells; it is capable of methylating in vitro homologous and heterologous DNA. Hence, DNA in bacterial cells can be partially undermethylated. This enzyme methylates cytosine residues in native and deneaturated DNA in the same nucleotide sequences. As compared to the native DNA, the denaturated DNA is indicative of a decrease in the level of methylation of adenine, rather than cytosine residues. Specificity of methylation of cytosine residues in vitro and in vivo does not depend on the nature of substrate DNA (calf thymus, Pseudomonas aeruginosa etc.). DNA-methylases of different variants of Bac. brevis (R, S, P+, P-) methylate cytosine residues in the same nucleotide sequences. It means that specificity of methylation of DNA cytosine residues in the cells of different variants of Bac. brevis is the same.     

Characterization and cloning of MwoI (GCN7GC), a new type-II restriction-modification system from Methanobacterium wolfei

R.MwoI, a type-II restriction enzyme with the new specificity 5'-GCN7GC-3', was found in extracts of the thermophilic archaebacterium, Methanobacterium wolfei. R.MwoI cleaves duplex DNA producing fragments with 3-nt, 3'-terminal extensions, thus: GCN5/N2GC. The genes coding for the MwoI restriction and modification enzymes were cloned into Escherichia coli on the plasmid vector pBR322. The clones synthesize a low level of R.MwoI endonuclease. The plasmids display incomplete MwoI-specific modification, suggesting that the clones synthesize a low level of the M.MwoI methyltransferase, too.     

Inference of relationships in the 'twilight zone' of homology using a combination of bioinformatics and site-directed mutagenesis: a case study of restriction endonucleases Bsp6I and PvuII

Thus far, identification of functionally important residues in Type II restriction endonucleases (REases) has been difficult using conventional methods. Even though known REase structures share a fold and marginally recognizable active site, the overall sequence similarities are statistically insignificant, unless compared among proteins that recognize identical or very similar sequences. Bsp6I is a Type II REase, which recognizes the palindromic DNA sequence 5′GCNGC and cleaves between the cytosine and the unspecified nucleotide in both strands, generating a double-strand break with 5′-protruding single nucleotides. There are no solved structures of REases that recognize similar DNA targets or generate cleavage products with similar characteristics. In straightforward comparisons, the Bsp6I sequence shows no significant similarity to REases with known structures. However, using a fold-recognition approach, we have identified a remote relationship between Bsp6I and the structure of PvuII. Starting from the sequence–structure alignment between Bsp6I and PvuII, we constructed a homology model of Bsp6I and used it to predict functionally significant regions in Bsp6I. The homology model was supported by site-directed mutagenesis of residues predicted to be important for dimerization, DNA binding and catalysis. Completing the picture of sequence–structure–function relationships in protein superfamilies becomes an essential task in the age of structural genomics and our study may serve as a paradigm for future analyses of superfamilies comprising strongly diverged members with little or no sequence similarity.     

Sse8647I, a new type II restriction endonuclease from a Streptomyces species cutting at 5'-AG/GWCCT-3'.

We isolated and characterized a new type II restriction endonuclease which recognizes the palindromic heptanucleotide sequence 5'-AGGWCCT-3' and cleaves double-stranded DNA after the first G in the sequence from a microorganism belonging to Streptomyces species. This enzyme cleaves adenovirus 2 DNA at eight sites, but does not cleave lambda phage, pBR322, pUC18 and 19, M13mp18 and 19, SV40, ColE1 and phi X174 DNAs.     

R.KpnI, an HNH superfamily REase, exhibits differential discrimination at non-canonical sequences in the presence of Ca2+ and Mg2+

KpnI REase recognizes palindromic sequence, GGTAC↓C, and forms complex in the absence of divalent metal ions, but requires the ions for DNA cleavage. Unlike most other REases, R.KpnI shows promiscuous DNA cleavage in the presence of Mg²⁺. Surprisingly, Ca²⁺ suppresses the Mg²⁺-mediated promiscuous activity and induces high fidelity cleavage. To further analyze these unique features of the enzyme, we have carried out DNA binding and kinetic analysis. The metal ions which exhibit disparate pattern of DNA cleavage have no role in DNA recognition. The enzyme binds to both canonical and non-canonical DNA with comparable affinity irrespective of the metal ions used. Further, Ca²⁺-imparted exquisite specificity of the enzyme is at the level of DNA cleavage and not at the binding step. With the canonical oligonucleotides, the cleavage rate of the enzyme was comparable for both Mg²⁺- and Mn²⁺-mediated reactions and was about three times slower with Ca²⁺. The enzyme discriminates non-canonical sequences poorly from the canonical sequence in Mg²⁺-mediated reactions unlike any other Type II REases, accounting for the promiscuous behavior. R.KpnI, thus displays properties akin to that of typical Type II REases and also endonucleases with degenerate specificity in its DNA recognition and cleavage properties.     

S-stereoselective piperazine-2-tert-butylcarboxamide hydrolase from Pseudomonas azotoformans IAM 1603 is a novel L-amino acid amidase.

An amidase acting on (R,S)-piperazine-2-tert-butylcarboxamide was purified from Pseudomonas azotoformans IAM 1603 and characterized. The enzyme acted S-stereoselectively on (R,S)-piperazine-2-tert-butylcarboxamide to yield (S)-piperazine-2-carboxylic acid. N-terminal and internal amino acid sequences of the enzyme were determined. The gene encoding the S-stereoselective piperazine-2-tert-butylcarboxamide amidase was cloned from the chromosomal DNA of the strain and sequenced. Analysis of 2.1 kb of genomic DNA revealed the presence of two ORFs, one of which (laaA) encodes the amidase. This enzyme, LaaA is composed of 310 amino acid residues (molecular mass 34 514 Da), and the deduced amino acid sequence exhibits significant similarity to hypothetical and functionally characterized proline iminopeptidases from several bacteria. The laaA gene modified in the nucleotide sequence upstream from its start codon was overexpressed in Escherichia coli. The activity of the recombinant LaaA enzyme in cell-free extracts of E. coli was 13.1 units.mg(-1) with l-prolinamide as substrate. This enzyme was purified to electrophoretic homogeneity by ammonium sulfate fractionation and two column chromatography steps. On gel-filtration chromatography, the enzyme appeared to be a monomer with a molecular mass of 32 kDa. It had maximal activity at 45 degrees C and pH 9.0, and was completely inactivated in the presence of phenylhydrazine, Zn2+, Ag+, Cd2+ or Hg2+. LaaA had hydrolyzing activity toward L-amino acid amides such as L-prolinamide, L-proline-p-nitroanilide, L-alaninamide and L-methioninamide, but did not act on the peptide substrates for the proline iminopeptidases despite their sequence similarity to LaaA. The enzyme also acted S-stereoselectively on (R,S)-piperidine-2-carboxamide, (R,S)-piperazine-2-carboxamide and (R,S)-piperazine-2-tert-butylcarboxamide. Based on its specificity towards L-amino acid amides, the enzyme was named L-amino acid amidase. E. coli transformants overexpressing the laaA gene could be used for the S-stereoselective hydrolysis of (R,S)-piperazine-2-tert-butylcarboxamide.     

DNA sequence preferences of GAL4 and PPR1: how a subset of Zn2 Cys6 binuclear cluster proteins recognizes DNA.

Biophysical and genetic experiments have defined how the Saccharomyces cerevisiae protein GAL4 and a subset of related proteins recognize specific DNA sequences. We assessed DNA sequence preferences of GAL4 and a related protein, PPR1, in an in vitro DNA binding assay. For GAL4, the palindromic CGG triplets at the ends of the 17-bp recognition site are essential for tight binding, whereas the identities of the internal 11 bp are much less important, results consistent with the GAL4-DNA crystal structure. Small reductions in affinity due to mutations at the center-most 5 bp are consistent with the idea that an observed constriction in the minor groove in the crystalline GAL4-DNA complex is sequence dependent. The crystal structure suggests that this sequence dependence is due to phosphate contacts mediated by arginine 51, as part of a network of hydrogen bonds. Here we show that the mutant protein GAL4(1-100)R51A fails to discriminate sites with alterations in the center of the site from the wild-type site. PPR1, a relative of GAL4, also recognizes palindromic CGG triplets at the ends of its 12-bp recognition sequence. The identities of the internal 6 bp do not influence the binding of PPR1. We also show that the PPR1 site consists of a 12-bp duplex rather than 16 bp as reported previously: the two T residues immediately 5' to the CGG sequence in each half site, although highly conserved, are not important for binding by PPR1. Thus, GAL4 and PPR1 share common CGG half sites, but they prefer DNA sequences with the palindromic CGG separated by the appropriate number of base pairs, 11 for GAL4 and 6 for PPR1.     

A view of consecutive binding events from structures of tetrameric endonuclease SfiI bound to DNA

Many reactions in cells proceed via the sequestration of two DNA molecules in a synaptic complex. SfiI is a member of a growing family of restriction enzymes that can bind and cleave two DNA sites simultaneously. We present here the structures of tetrameric SfiI in complex with cognate DNA. The structures reveal two different binding states of SfiI: one with both DNA-binding sites fully occupied and the other with fully and partially occupied sites. These two states provide details on how SfiI recognizes and cleaves its target DNA sites, and gives insight into sequential binding events. The SfiI recognition sequence (GGCCNNNN[downward arrow]NGGCC) is a subset of the recognition sequence of BglI (GCCNNNN[downward arrow]NGGC), and both enzymes cleave their target DNAs to leave 3-base 3' overhangs. We show that even though SfiI is a tetramer and BglI is a dimer, and there is little sequence similarity between the two enzymes, their modes of DNA recognition are unusually similar.     

SruI restriction endonuclease from Selenomonas ruminantium

Abstract Sru I, specific restriction endonuclease, has been characterized from Selenomonas ruminantium isolated from the rumen of fallow deer. Results from the study demonstrate that S. ruminantium 18D possesses a type II restriction endonuclease, which recognizes the sequence 5'-TTT↓AAA-3'. The recognition sequence of Sru I was identified using digestions on pBR322, pBR328, pUC18, M13mp18RF, pACYC184 and λDNA. The cleavage patterns obtained were compared with computer-derived data. Sru I recognises the palindromic hexanucleotide sequence and cleaves DNA after the third T in the sequence, producing blunt ends. The purification and characterization of restriction endonuclease Sru I presented here is the first described for Selenomonas ruminantium spp. and demonstrates that this microorganism pocesses a DNA-cleaving enzyme with the same specificity as Dra I or Aha III.