Efficient methodology for the cyclization of linear peptide libraries via intramolecular S-alkylation using Multipin solid phase peptide synthesis.

Methodology is described here for the efficient parallel synthesis and cyclization of linear peptide libraries using intramolecular S-alkylation chemistry in combination with Multipin solid phase peptide synthesis (Multipin SPPS). The effective use of this methodology was demonstrated with the synthesis of a 72-member combinatorial library of cyclic thioether peptide derivatives of the conserved four-residue structural motif DD/EXK found in the active sites of the five crystallographically defined orthodox type II restriction endonucleases, EcoRV, EcoRI, PvuII, BamHI and BglI.     

Physical map of Pseudomonas aeruginosa phage phi kF77 genome. Localization of sites sensitive to restriction endonucleases

A physical map of the P. aeruginosa bacteriophage phi kF77 has been constructed using the restriction endonucleases SalI, HindIII, EcoRI, EcoRV, MuI, XbaI, ClaI. The phi kF77 DNA is resistant to cleavage by the restriction endonucleases BamHI, BglII, HpaI, PstI, PvuII, SmaI, XhoI.     

Modulating restriction endonuclease activities and specificities using neutral detergents

It is well known that type II restriction enzyme activities and specificities can be modulated by altering solution conditions. The addition of co-solvents such as dimethyl sulfoxide (DMSO), alcohols and polyols can promote star activity, which is the cleavage of non-cognate sequences. While neutral detergents are often used to control protein aggregation, little is known about the effect of neutral detergents on restriction enzyme activities and specificities. We report here that BamHI, BglI, BglII, EcoRI, EcoRV, HindIII, MluI, PvuII, SalI and XhoI restriction endonucleases are remarkably tolerant of high concentrations of neutral detergents Triton X-100, CHAPS and octyl glucoside. In most cases, lambda DNA cleavage rates were comparable to those observed in the absence of detergent. Indeed, the specific activities of SalI and XhoI were appreciably increased in the presence of Triton X-100. For all enzymes active in the presence of detergents, sequence specificity toward lambda DNA was not compromised. Assays of star cleavage of pUC18 by EcoRI, PvuII and BamHI endonucleases in equimolar concentrations of Triton X-100 and sucrose revealed reduced star activity in the detergent relative to the sucrose co-solvent. Interestingly, under star activity-promoting conditions, PvuII endonuclease displayed greater fidelity in Triton X-100 than in conventional buffer. Taken altogether, these results suggest that in some cases, neutral detergents can be used to manipulate restriction endonuclease reaction rates and specificities.     

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.     

Immobilization of restrictases

The search for optimal variants of restriction endonucleases immobilization was begun recently. For some enzymes immobilization was successful due to the presence of covalent bonds on CNBr-sepharose (EcoRI, BamHI, HindIII, TaqI, PaeI, SalI, PvuII). For the enzymes EcoRI, BamHI and HindIII it was due to hydrophobic interaction with triethyl-agarose (triethyl-triphenylmethane). The high yield (up to 80%) of enzymatic activity has been obtained for small number of restriction endonucleases. In the experiments of several amino acid residues modification and immobilization of restriction endonucleases the participation of lysine, arginine, glutamic acid and SH- or S-S-groups in the catalysis and (or) binding of these enzymes with DNA has been shown. The restriction endonucleases immobilization experiments and research of enzymes active centre enrich each other and are very interesting for their use in molecular biology and deepening our knowledge of protein-nucleic interactions.     

Structure of PvuII endonuclease with cognate DNA.

We have determined the structure of PvuII endonuclease complexed with cognate DNA by X-ray crystallography. The DNA substrate is bound with a single homodimeric protein, each subunit of which reveals three structural regions. The catalytic region strongly resembles structures of other restriction endonucleases, even though these regions have dissimilar primary sequences. Comparison of the active site with those of EcoRV and EcoRI endonucleases reveals a conserved triplet sequence close to the reactive phosphodiester group and a conserved acidic pair that may represent the ligands for the catalytic cofactor Mg2+. The DNA duplex is not significantly bent and maintains a B-DNA-like conformation. The subunit interface region of the homodimeric protein consists of a pseudo-three-helix bundle. Direct contacts between the protein and the base pairs of the PvuII recognition site occur exclusively in the major groove through two antiparallel beta strands from the sequence recognition region of the protein. Water-mediated contacts are made in the minor grooves to central bases of the site. If restriction enzymes do share a common ancestor, as has been proposed, their catalytic regions have been very strongly conserved, while their subunit interfaces and DNA sequence recognition regions have undergone remarkable structural variation.     

Genetic characteristics and physical organization of the R-plasmid pBS52 with a broad range of bacterial hosts

The genetic and physical data on Pseudomonas aeruginosa plasmid pBS52 coding for the resistances to ampicillin, streptomycin and sulfonamids have been obtained. This conjugative plasmid is transferable to a broad range of gram-negative bacterial hosts and compatible with the broad host-range plasmids from all known incompatibility groups. The plasmid size has been determined (38 Kb) and a physical map has been constructed using restriction endonucleases EcoRI, EcoRV, BamHI, BglII, PstI, PvuII, SalI, SlaI. The presence of a fragment, approximately 200 bp in size, which contains the sites for many of widely used restriction endonucleases is a characteristic feature of the plasmid pBS52.     

PvuII endonuclease contains two calcium ions in active sites

Restriction endonucleases differ in their use of metal cofactors despite having remarkably similar folds for their catalytic regions. To explore this, we have characterized the interaction of endonuclease PvuII with the catalytically incompetent cation Ca(2+). The structure of a glutaraldehyde-crosslinked crystal of the endonuclease PvuII-DNA complex, determined in the presence of Ca(2+) at a pH of approximately 6.5, supports a two-metal mechanism of DNA cleavage by PvuII. The first Ca(2+) position matches that found in all structurally examined endonucleases, while the second position is similar to that of EcoRV but is distinct from that of BamHI and BglI. The location of the second metal in PvuII, unlike that in BamHI/BglI, permits no direct interaction between the second metal and the O3' oxygen leaving group. However, the interactions between the DNA scissile phosphate and the metals, the first metal and the attacking water, and the attacking water and DNA are the same in PvuII as they are in the two-metal models of BamHI and BglI, but are distinct from the proposed three-metal or the two-metal models of EcoRV.     

Physical map of the Rhodobacter sphaeroides bacteriophage phi RsG1 genome and location of the prophage on the host chromosome.

We constructed a physical map of the 50-kilobase-pair (kb) DNA of the temperate Rhodobacter sphaeroides bacteriophage phi RsG1, with the relative positions of the cleavage sites for the nine restriction endonucleases KpnI, HindIII, XbaI, ClaI, BclI, EcoRV, EcoRI, BglII, and BamHI indicated. Using biotinylated phi RsG1 DNA as a probe in hybridization studies, we detected homologies with virus DNA and fragments of restriction endonuclease-digested host chromosomal DNA but not with plasmid DNA. This indicates that the prophage is integrated into the host chromosome. In addition, the use of specific probes such as the 10.4-kb BglII A fragment and the 2.65-kb BamHI H fragment allowed the determination of the position of phage attachment site (attP).     

Identification of the metal-binding sites of restriction endonucleases by Fe2+-mediated oxidative cleavage

Fenton chemistry [Fenton (1894) J. Chem. Soc. 65, 899-910] techniques were employed to identify the residues involved in metal binding located at the active sites of restriction endonucleases. This process uses transition metals to catalytically oxidize the peptide linkage that is in close proximity to the amino acid residues involved in metal ligation. Fe2+ was used as the redox-active transition metal. It was expected that Fe2+ would bind to the endonucleases at the Mg2+-binding site [Liaw et al. (1993) Biochemistry 32, 7999-4003; Ermácora et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6383-6387; Soundar and Colman (1993) J. Biol. Chem. 268, 5264-5271; Wei et al. (1994) Biochemistry 33, 7931-7936; Ettner et al. (1995) Biochemistry 34, 22-31; Hlavaty and Nowak (1997) Biochemistry 36, 15515-15525). Fe2+-mediated oxidation was successfully performed on TaqI endonulease, suggesting that this approach could be applied to a wide array of endonucleases [Cao and Barany (1998) J. Biol. Chem. 273, 33002-33010]. The restriction endonucleases BamHI, FokI, BglI, BglII, PvuII, SfiI, BssSI, BsoBI, EcoRI, EcoRV, MspI, and HinP1I were subjected to oxidizing conditions in the presence of Fe2+ and ascorbate. All proteins were inactivated upon treatment with Fe2+ and ascorbate. BamHI, FokI, BglI, BglII, PvuII, SfiI, BssSI, and BsoBI were specifically cleaved upon treatment with Fe2+/ascorbate. The site of Fe2+/ascorbate-induced protein cleavage for each enzyme was determined. The Fe2+-mediated oxidative cleavage of BamHI occurs between residues Glu77 and Lys78. Glu77 has been shown by structural and mutational studies to be involved in both metal ligation and catalysis [Newman et al. (1995) Science 269, 656-663; Viadiu and Aggarwal (1998) Nat. Struct. Biol. 5, 910-916; Xu and Schildkraut (1991) J. Biol. Chem. 266, 4425-4429]. The sites of Fe2+/ascorbate-induced cleavage for PvuII, FokI, BglI, and BsoBI agree with the metal-binding sites identified in their corresponding three-dimensional structures or from mutational studies [Cheng et al. (1994) EMBO J. 13, 3297-3935; Wah et al. (1997) Nature 388, 97-100; Newman et al. (1998) EMBO J. 17, 5466-5476; Ruan et al. (1997) Gene 188, 35-39]. The metal-binding residues of BglII, SfiI, and BssSI are proposed based on amino acid sequencing of their Fe2+/ascorbate-generated cleavage fragments. These results suggest that Fenton chemistry may be a useful methodology in identifying amino acids involved in metal binding in endonucleases.     

Restriction enzyme analysis of Bacillus subtilis bacteriophage phi 105 DNA

The recognition sites on phi 105 DNA for the restriction endonucleases EcoRI, Bg/II, SmaI, KpnI, SstI, SalI, XhoI, NcoI, PstI, HindIII, ClaI, EcoRV and MluI have been mapped. The sites for EcoRI are shown to be different from those published earlier. The DNA from phi 105 contains no recognition sites for the endonucleases BamHI and XbaI.     

New shuttle-type vectors for cloning in Escherichia coli and Agrobacterium tumefaciens

The vectors capable of replication in Escherichia coli and Agrobacterium tumefaciens have been constructed on the basis of the plasmid pUB5502. The constructed vectors pVA12, pVA12-2, pVA12-4 contain the mini-replicon and trimethoprim resistance gene (Tp) of a broad host-range plasmid R388 (IncW). The pVA12 vector (8.8 kb) has been constructed by insertion of a kanamycin resistance gene (Km) from the plasmid pUC-4K into a Psti site. It possesses 7 unique restriction sites for XhoI, SmaI, PvuI, PvuII, HindIII, EcoRI, BamHI and the markers for kanamycin and trimethoprim resistance (Km and Tp). The pVA12-2 and pVA12-4 vectors were obtained as a result of changing of the PvuII-EcoI fragment of pVA12 carrying the Tp gene for the PvuII-EcoRI fragment of pBR322 carrying the Tc gene. These plasmids have the same size of 9.7 kb and 8 unique sites for restriction endonucleases XhoI, SmaI, PvuI, PvuII, EcoRI, EcoRV, SalI, BalI and Km and Tc genes. No difference has been registered between the two plasmids by restriction analysis, but pVA12-4 has the dramatically increased copy number in Escherichia coli cells. All three vectors are transferable to Agrobacterium tumefaciens with the same frequencies by transformation or conjugation and do not affect the oncogenicity of pTi.     

Restriction site polymorphism in rDNA repeat unit ofHordeum vulgare for some restriction endonucleases

Restriction fragment length polymorphism in rDNA repeat unit for eight restriction endonucleases was studied in two genotypes of barley (Hordeum vulgare L.). Both genotypes contained restriction sites for EcoRI, EcoRV, PvuII and Sau3239I in rDNA repeat unit and contained two different rDNA repeat unit length variants 9.7 kbp and 9.1 kbp.     

Induction of CAD gene amplification by restriction endonucleases in V79,B7 Chinese hamster cells

The restriction endonucleases PvuII, BamHI and EcoRI were tested for their ability to induce gene amplification in V79,B7 Chinese hamster cells. The results indicate that treatment with these enzymes efficiently increases the frequency of clones resistant to N-phosphonacetyl-L-aspartate, indicating induction of CAD gene amplification.     

Restriction endonuclease cleavage maps of the ampicillin transposons Tn2601 and Tn2602

This paper reports the cleavage maps of ampicillin transposons Tn2601 and Tn2602, for restriction endonucleases BamHI, PvuII, AvaI, HincII, and HaeII. Both of the transposons are very similar to the well-known ampicillin transposon Tn3 in size, endonuclease cleavage sites, and possession of a short inverted repeat sequence at both ends. A slight difference in the cleavage pattern among these three transposons was observed in the region around the BamHI site which was assumed to be a part of the repressor gene for transposition.     

Protein stability indicates divergent evolution of PD-(D/E)XK type II restriction endonucleases

Type II restriction endonucleases recognize 4-8 base-pair-long DNA sequences and catalyze their cleavage with remarkable specificity. Crystal structures of the PD-(DE)XK superfamily revealed a common alpha/beta core motif and similar active site. In contrast, these enzymes show little sequence similarity and use different strategies to interact with their substrate DNA. The intriguing question is whether this enzyme family could have evolved from a common origin. In our present work, protein structure stability elements were analyzed and compared in three parts of PD-(DE)XK type II restriction endonucleases: (1) core motif, (2) active-site residues, and (3) residues playing role in DNA recognition. High correlation was found between the active-site residues and those stabilization factors that contribute to preventing structural decay. DNA recognition sites were also observed to participate in stabilization centers. It indicates that recognition motifs and active sites in PD-(DE)XK type II restriction endonucleases should have been evolutionary more conserved than other parts of the structure. Based on this observation it is proposed that PD-(DE)XK type II restriction endonucleases have developed from a common ancestor with divergent evolution.     

Linkage Map of Escherichia coli K-12, Edition 10: The Physical Map

A physical map, EcoMap10, of the now completely sequenced Escherichia coli chromosome is presented. Calculated genomic positions for the eight restriction enzymes BamHI, HindIII, EcoRI, EcoRV, BglI, KpnI, PstI, and PvuII are depicted. Both sequenced and unsequenced Kohara/Isono miniset clones are aligned to this calculated restriction map. DNA sequence searches identify the precise locations of insertion sequence elements and repetitive extragenic palindrome clusters. EcoGene10, a revised set of genes and functionally uncharacterized open reading frames (ORFs), is also depicted on EcoMap10. The complete set of unnamed ORFs in EcoGene10 are assigned provisional names beginning with the letter “y” by using a systematic nomenclature.     

Restriction maps and restriction fragment length polymorphisms of the human 21-hydroxylase genes

Restriction maps were constructed for the two human 21-hydroxylase genes (21-OHA and 21-OHB) by using DNA from subjects homozygous for a deletion of each gene. Comparing the patterns of these two genes, a KpnI restriction site occurred in the 21-OHA gene in place of a TaqI site in the 21-OHB gene about 1-kb from the 5' end of the gene, and an extra EcoRI site was located 500 bp 5' to the common EcoRI site. The DNA of fourteen unrelated normal subjects was digested with nine restriction endonucleases (AccI, BamHI, BgIII, EcoRI, HindIII, KpnI, MspI, SacI and TaqI). Restriction fragment length polymorphisms were found with EcoRI, HindIII and AccI that resulted from polymorphic endonuclease sites outside the genes.     

The detailed physical map of the temperate phage 16-3 of Rhizobium meliloti 41

Restriction cleavage maps for enzymes EcoRI, BamHI, PstI, PvuII, XbaI and EcoRV of Rhizobium meliloti temperate phage 16-3 have been established. Together with the earlier maps (HindIII, KpnI, HpaI, BglII) 98 restriction sites, 'evenly' distributed, have been mapped along the phage genome, including the so far unmarked silent region of the chromosome. All the restriction maps have been fitted to each other by computer optimalization. Beyond for conventional techniques a computer program (PMAP) for physical mapping of linear DNA has been employed which made the experimentation, in several cases, extremely efficient.     

Restriction endonuclease analysis of mitochondrial DNA of three farm animal species: Cattle, sheep and goat

Five restriction endonucleases (HindIII, BgIII, EcoRI, EcoRV and BamHI) were employed to analyse mitochondrial DNA of cattle, sheep and goat. The results showed completely different restriction patterns of mtDNA among the three bovid species. A total of 11, 16, and 17 restriction fragments in cattle, sheep and goat respectively, were detected by the five restriction endonucleases. Average total sizes of mtDNA of cattle, sheep and goat were found to be 16.49 ± 0.18, 16.30 ± 0.25 and 16.44 ± 0.08 kb, respectively. The mtDNA cleavage patterns were identical for all seven individuals belonging to two cattle breeds and for 10 individuals from one sheep breed.