Determination of substrate specificity of restriction endonuclease FauI

The recognition sequence and cleavage point of restriction endonuclease FauI have been determined as 5'-CCCGC(4/6). Not being isoschisomer of any known restriction endonuclease, this enzyme may be used in genetic engineering.     

Isolation of BsoBI restriction endonuclease variants with altered substrate specificity

BsoBI is a thermophilic restriction endonuclease that cleaves the degenerate DNA sequence C/PyCGPuG (where/=the cleavage site and Py=C or T, Pu=A or G). In the BsoBI-DNA co-crystal structure the D246 residue makes a water-mediated hydrogen bond to N6 of the degenerate base adenine and was proposed to make a complementary bond to O6 of the alternative guanine residue. To investigate the substrate specificity conferred by D246 and to potentially alter BsoBI specificity, the D246 residue was changed to the other 19 amino acids. Variants D246A, D246C, D246E, D246R, D246S, D246T, and D246Y were purified and their cleavage activity determined. Variants D246A, D246S, and D246T display 0.2% to 0.7% of the wild-type cleavage activity. However, the substrate specificity of the three variants is altered significantly. D246A, D246S, and D246T cleave CTCGAG sites poorly. In filter binding assays using oligonucleotides, wild-type BsoBI shows almost equal affinity for CTCGAG and CCCGGG sites. In contrast, the D246A variant shows 70-fold greater binding affinity for the CCCGGG substrate. Recycled mutagenesis was carried out on the D246A variant, and revertants with enhanced activity were isolated by their dark blue phenotype on a dinD Colon, two colons lacZ DNA damage indicator strain. Most of the amino acid substitutions present within the revertants were located outside the DNA-protein interface. This study demonstrates that endonuclease mutants with altered specificity and non-lethal activity can be evolved towards more active variants using a laboratory evolution strategy.     

Directed evolution of restriction endonuclease BstYI to achieve increased substrate specificity

Restriction endonucleases have proven to be especially resistant to engineering altered substrate specificity, in part, due to the requirement of a cognate DNA methyltransferase for cellular DNA protection. The thermophilic restriction endonuclease BstYI recognizes and cleaves all hexanucleotide sequences described by 5'-R GATCY-3' (where R=A or G and Y=C or T). The recognition of a degenerate sequence is a relatively common feature of the more than 3000 characterized restriction endonucleases. However, very little is known concerning substrate recognition by such an enzyme. Our objective was to investigate the substrate specificity of BstYI by attempting to increase the specificity to recognition of only AGATCT. By a novel genetic selection/screening process, two BstYI variants were isolated with a preference for AGATCT cleavage. A fundamental element of the selection process is modification of the Escherichia coli host genomic DNA by the BglII N4-cytosine methyltransferase to protect AGATCT sites. The amino acid substitutions resulting in a partial change of specificity were identified and combined into one superior variant designated NN1. BstYI variant NN1 displays a 12-fold preference for cleavage of AGATCT over AGATCC or GGATCT. Moreover, cleavage of the GGATCC sequence is no longer detected. This study provides further evidence that laboratory evolution strategies offer a powerful alternative to structure-guided protein design.     

Purification and substrate specificity of a T4 phage intron-encoded endonuclease.

The T4 phage td intron-encoded endonuclease (I-Tev I) cleaves the intron-deleted td gene (td delta I) 23 nucleotides upstream of the intron insertion site on the noncoding strand and 25 nucleotides upstream of this site on the coding strand, to generate a 2-base hydroxyl overhang in the 3' end of each DNA strand. I-Tev I-157, a truncated form in which slightly more than one third (88 residues) of the endonuclease is deleted, was purified to homogeneity and shown to possess endonuclease activity similar to that of I-TEV I, the full-length enzyme (245 residues). The minimal length of the td delta I gene that was cleaved by I-Tev I and I-Tev I-157 has been determined to be exactly 39 basepairs, from -27 (upstream in exon1) to +12 (downstream in exon2) relative to the intron insertion site. Similar to the full-length endonuclease, I-Tev I-157 cuts the intronless thymidylate synthase genes from such diverse organisms as Escherichia coli, Lactobacillus casei and the human. The position and nature of the in vitro endonucleolytic cut in these genes are homologous to those in td delta I. Point mutational analysis of the td delta I substrate based on the deduced consensus nucleotide sequence has revealed a very low degree of specificity on either side of the cleavage site, for both the full-length and truncated I-TEV I.     

Purification, properties and specificity of the restriction endonuclease from Bacillus stearothermophilus.

The restriction endonuclease BstI was purified from 70kg of Bacillus stearothermophilus. The final product is at least 97% pure as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; this major protein species co-migrates with the enzyme activity on native polyacrylamide-gel electrophoresis and isoelectric focusing. Pure restriction endonuclease BstI has a subunit mol.wt. of 26,000 and is probably a loosely associated dimer. The enzyme shows maximum activity at pH values between 7 and 9.5, and in the presence of 0.5-2mM-Mg2+. NaCl inhibits the restriction enzyme activity. Restriction endonuclease BstI cleaves DNA in a position identical with that cleaved by endonuclease BamHI (for Bacillus amyloliquefaciens), i.e.: (formula: see text). In the presence of high concentrations of enzyme, DNA cleavage occurs at secondary sites. This side-specificity is enhanced by the addition of glycerol. Preliminary studies indicate that these sites are of the type: (formula: see text).     

Substrate specificity of restriction endonuclease SfeI

The recognition sequence and cleavage site C decreases TRYAG of a new restriction endonuclease SfeI have been determined.     

Substrate specificity of restriction endonuclease VspI

The recognition sequence and cleavage point of restriction endonuclease VspI have been determined as 5'-AT decreases TAAT. This enzyme is not isoschizomer of any known restriction endonucleases. DNA pBR322 contains a single VspI recognition sequence in position 3539. Therefore this enzyme may be used for cloning DNA in the VspI site in AmpR-gene of pBR322.     

Relaxed specificity of the EcoRV restriction endonuclease

The EcoRV restriction endonuclease normally shows a high specificity for its recognition site on DNA, GATATC. In standard reactions, it cleaves DNA at this site several orders of magnitude more readily than at any alternative sequence. But in the presence of dimethyl sulphoxide and at high pH, the EcoRV enzyme cleaves DNA at several sites that differ from its recognition site by one nucleotide. Of the 18 (3 X 6) possible sequences that differ from GATATC by one base, all were cleaved readily except for the following 4 sites: TATATC, CATATC, GATATA and GATATG. However, two of the sites that could be cleaved by EcoRV in the presence of dimethyl sulphoxide, GAGATC and GATCTC, were only cleaved on DNA that lacked dam methylation: both contain the sequence GATC, the recognition site for the dam methylase of Escherichia coli.     

Substrate specificity of restriction endonuclease Eco781

The recognition sequence and cleavage point of restriction endonuclease Eco781 have been determined as 5'-GGCGCC-. There are several known enzymes recognizing the same sequence, although the prototype NarI and isoschizomers NdaI and NunII cleave the substrate to produce 5'-protruding ends, whereas cleavage with isoschizomer BbeI results in 3'-protruding ends. Therefore, restrictase Eco78I, generating flush ends, may be regarded as an enzyme with new specificity among the restriction endonucleases recognizing the 5'-GGCGCC-sequence.     

Purification and characterization of the FokI restriction endonuclease

The restriction endonuclease FokI from Flavobacterium okeanokoites was purified to homogeneity. Based on gel filtration, sedimentation and sodium dodecyl sulfate-polyacrylamide-gel electrophoresis, the following properties of the enzyme were determined: FokI exists in one active monomeric form, and has an Mr of 64-65.4 x 10(3).FokI is a strongly basic protein with an isoelectric point of 9.4. The enzyme exhibits restriction activity in the pH range 5.0 to 10.5 (maximum level at pH 7.0-8.5) and its divalent cation requirement is satisfied not only by Mg2+, but also by Co2+, Mn2+, Ni2+, Cd2+, Zn2+ and Fe2+.     

Purification and crystallization of the EcoRV restriction endonuclease

The type II restriction endonuclease EcoRV purified from a genetically engineered, overproducing strain has been crystallized. Four crystal forms all obtained by precipitation with polyethylene glycol 4000 have been characterized. Two of these are suitable for high resolution structure analysis. Both are orthorhombic, have space group P2(1)2(1)2(1) and have similar unit cell dimensions of a = 58.2 A, b = 71.7 A, c = 130.6 A (form A) and a = 59.9 A, b = 74.5 A, c = 121.8 A (form B). They diffract to about 2A resolution and appear to have one dimer of 2 X 29,000 daltons in the asymmetric unit.     

Purification of the restriction endonuclease PalI.

The restriction endonuclease PalI was purified from Providencia alcalifaciens 1650-fold with a yield of 33%. The purified protein moved as a single band upon polyacrylamide gel electrophoresis. When this was carried out in the presence of sodium dodecyl sulfate, a molecular weight of 31,000 was obtained for PalI. Gel filtration through Sephacryl S200 gave molecular weights ranging from 44,000 to 53,000 when 58 to 1870 ng/ml enzyme were used, respectively. Other properties of the enzyme are described.     

Increasing cleavage specificity and activity of restriction endonuclease KpnI

Restriction enzyme KpnI is a HNH superfamily endonuclease requiring divalent metal ions for DNA cleavage but not for binding. The active site of KpnI can accommodate metal ions of different atomic radii for DNA cleavage. Although Mg²⁺ ion higher than 500 μM mediates promiscuous activity, Ca²⁺ suppresses the promiscuity and induces high cleavage fidelity. Here, we report that a conservative mutation of the metal-coordinating residue D148 to Glu results in the elimination of the Ca²⁺-mediated cleavage but imparting high cleavage fidelity with Mg²⁺. High cleavage fidelity of the mutant D148E is achieved through better discrimination of the target site at the binding and cleavage steps. Biochemical experiments and molecular dynamics simulations suggest that the mutation inhibits Ca²⁺-mediated cleavage activity by altering the geometry of the Ca²⁺-bound HNH active site. Although the D148E mutant reduces the specific activity of the enzyme, we identified a suppressor mutation that increases the turnover rate to restore the specific activity of the high fidelity mutant to the wild-type level. Our results show that active site plasticity in coordinating different metal ions is related to KpnI promiscuous activity, and tinkering the metal ion coordination is a plausible way to reduce promiscuous activity of metalloenzymes.     

The SalGI restriction endonuclease. Purification and properties

The type II restriction endonuclease SalGI has been purified to near homogeneity. At least 80% of the protein remaining after the final stage of the preparation is SalGI restriction endonuclease; no contaminating nucleases remain detectable. The principal form of the protein under both native and denaturing conditions is a monomer of M(r) about 29000. The optimal conditions for both enzyme stability and enzyme activity have been determined.     

The SalGI restriction endonuclease. Enzyme specificity.

We have analysed the kinetics of DNA cleavage in the reaction between the SalGI restriction endonuclease and plasmid pMB9. This reaction is subject to competitive inhibition by DNA sequences outside the SalGI recognition site; we have determined the Km and Vmax. for the reaction of this enzyme at its recognition site and the KI for its interaction at other DNA sequences. We conclude that the specificity of DNA cleavage by the enzyme is only partly determined by the discrimination it shows for binding at its recognition sequence compared with binding to other DNA sequences.     

Purification of restriction endonuclease XcyI from Xanthomonas cyanopsidis

A new Type II restriction endonuclease XcyI, purified from Xanthomonas cyanopsidis 13D5, is an isoschizomer of SmaI and XmaI that cleaves at the nucleotide sequence 5'-C decreases CCGGG-3' of double-stranded DNA. The single restriction activity present in this strain permits rapid purification of 8000 units of cleavage activity from 10 g of freshly harvested cells. The resulting XcyI preparation is free of contaminating nuclease activities that interfere with in vitro manipulation of DNA.     

Massively parallel determination and modeling of endonuclease substrate specificity

We describe the identification and characterization of novel homing endonucleases using genome database mining to identify putative target sites, followed by high throughput activity screening in a bacterial selection system. We characterized the substrate specificity and kinetics of these endonucleases by monitoring DNA cleavage events with deep sequencing. The endonuclease specificities revealed by these experiments can be partially recapitulated using 3D structure-based computational models. Analysis of these models together with genome sequence data provide insights into how alternative endonuclease specificities were generated during natural evolution.