In vitro sodium bisulfite mutagenesis of restriction endonuclease recognition sites

Sodium bisulfite treatment of single-stranded DNA deaminates exposed cytosine residues to form uracil, resulting in cytosine-to-thymidine transition mutations following DNA replication. We have used this reaction in vitro to destroy the recognition sequences for the restriction endonucleases HindIII and XmaI in the aminoglycoside 3'-phosphotransferase I coding region of plasmid pUC4K. This procedure should be applicable to the mutation of any recognition sequence of restriction endonucleases which generate cytosine-containing single-stranded ends. The possibility of mutagenesis of restriction sites to generate stop codons in coding regions is discussed.     

REF Select: expert system software for selecting restriction endonucleases for restriction endonuclease fingerprinting

REF Select, expert system software, has been developed to assist in the selection of optimal restriction endonucleases for restriction endonuclease fingerprinting (REF), a method for rapid and sensitive mutation screening of long DNA segments (1-2 kb). The REF method typically involves six separate digestions with up to two restriction endnonucleases used in each digestion. If done manually, performing a comprehensive review of the large number of possible sets of restriction endonucleases that could be used (over 10(19) in the example presented here) and making an optimal choice is not feasible. Furthermore, the typical nonoptimal manual selection takes approximately 8 h by someone experienced with REF. REF Select enables a comprehensive review of the possible sets and a consistent, objective and fast selection of an optimal set by using a two-step strategy: the selection of sets that meet specific constraints, which is followed by a ranking of those sets by an optimality score. Based on our experience with REF, we chose default selection and ranking parameters to help the user get started quickly. These parameters form a knowledge base that can be customized and then saved by the user. In conclusion, REF Select facilitates the general application of REF by serving as an expert system for the selection of optimal restriction endonucleases. We demonstrated REF Select using an example segment from the human p53 gene.     

Selective protection of restriction endonuclease sites

A difficulty that is encountered when attempting to insert a PCR-amplified product or DNA fragment of interest into a particular vector is the presence within the insert of one or more internal restriction endonuclease (RE) sites identical to those selected for the flanks of the insert. Our method circumvents this problem by partially protecting internal RE sites while flanking sites for the same RE are cleaved. The amplified product is first heat denatured in the presence of excess amounts of perfectly complementary oligonucleotides that can anneal to the flanks of the insert. The mixture is allowed to anneal and is subsequently digested with the appropriate endonucleases. This results in the cleavage of the flanking RE sites while digestion at the internal RE site is not efficient. The mixture is subsequently heat denatured and column purified to remove the oligonucleotides. The product is then allowed to anneal and can be used directly in a ligation reaction with the plasmid vector. This method facilitates the construction of recombinant molecules by creating desired flanks while preserving internal RE sites.     

Codification and evolution of experimentally observed specific recognition sites for restriction enzymes on DNA

The list of published restriction endonucleases along with their substrates provides an excellent data base for the evaluation of the evolution and codification of the key elements for specific recognition sites on the DNA. In this paper the considerations will be limited to palindromic tetramer-, pentamer-, and hexamer-sequences. It is basically assumed that each base pair within these sequences has to be recognized by directionally unique bidentate hydrogen bonds either within the plane of the base pair or by bridging the appropriate H-bond donor/acceptor groups of the neighbouring bases of the same strand. Thus sequence specificity is mediated by twelve (eight) H-bonds, originating from the protein recognition modules. Besides a pronounced preference for GC base pairs expressed by their high frequency in the most abundant sequences, serving the need of maximal thermodynamic stability of the double helical substrates, it can also be shown that the stacking of consecutive bases within the recognition site sequences plays a major role in shaping the particular DNA/protein interface. Finally it will be demonstrated that the full set of sequences discussed in this paper can readily be derived by stepwise expanding the vocabulary of three simple tetrameric sequences by inserting single base pairs into the centre of a minimal sequence, thus creating all the published pentameric restriction sites, or by inserting/adding two GC base pairs in a palindromic way, thus creating the known multiplicity of hexameric sites.     

Design of endonuclease restriction sites into primers for PCR cloning

I present a software system PCRCLNG that facilitates the design of endonuclease restriction sites into the 5'-end of PCR primers. The product amplified using these primers can be directly cloned into vectors. The program estimates the annealing temperature for each primer and selects the primer pairs with comparable annealing temperature. Finally the software determines whether the PCR product can be cloned into the vector to generate in-frame gene fusion.     

Developing a programmed restriction endonuclease for highly specific DNA cleavage

Specific cleavage of large DNA molecules at few sites, necessary for the analysis of genomic DNA or for targeting individual genes in complex genomes, requires endonucleases of extremely high specificity. Restriction endonucleases (REase) that recognize DNA sequences of 4-8 bp are not sufficiently specific for this purpose. In principle, the specificity of REases can be extended by fusion to sequence recognition modules, e.g. specific DNA-binding domains or triple-helix forming oligonucleotides (TFO). We have chosen to extend the specificity of REases using TFOs, given the combinatorial flexibility this fusion offers in addressing a short, yet precisely recognized restriction site next to a defined triple-helix forming site (TFS). We demonstrate here that the single chain variant of PvuII (scPvuII) covalently coupled via the bifunctional cross-linker N-(gamma-maleimidobutryloxy) succinimide ester to a TFO (5'-NH2-[CH2](6 or 12)-MPMPMPMPMPPPPPPT-3', with M being 5-methyl-2'-deoxycytidine and P being 5-[1-propynyl]-2'-deoxyuridine), cleaves DNA specifically at the recognition site of PvuII (CAGCTG) if located in a distance of approximately one helical turn to a TFS (underlined) complementary to the TFO ('addressed' site: 5'-TTTTTTTCTCTCTCTCN(approximately 10)CAGCTG-3'), leaving 'unaddressed' PvuII sites intact. The preference for cleavage of an 'addressed' compared to an 'unaddressed' site is >1000-fold, if the cleavage reaction is initiated by addition of Mg2+ ions after preincubation of scPvuII-TFO and substrate in the absence of Mg2+ ions to allow triple-helix formation before DNA cleavage. Single base pair substitutions in the TFS prevent addressed DNA cleavage by scPvuII-TFO.     

Simultaneous binding of three recognition sites is necessary for a concerted plasmid DNA cleavage by EcoRII restriction endonuclease

According to the current paradigm type IIE restriction endonucleases are homodimeric proteins that simultaneously bind to two recognition sites but cleave DNA at only one site per turnover: the other site acts as an allosteric locus, activating the enzyme to cleave DNA at the first. Structural and biochemical analysis of the archetypal type IIE restriction enzyme EcoRII suggests that it has three possible DNA binding interfaces enabling simultaneous binding of three recognition sites. To test if putative synapsis of three binding sites has any functional significance, we have studied EcoRII cleavage of plasmids containing a single, two and three recognition sites under both single turnover and steady state conditions. EcoRII displays distinct reaction patterns on different substrates: (i) it shows virtually no activity on a single site plasmid; (ii) it yields open-circular DNA form nicked at one strand as an obligatory intermediate acting on a two-site plasmid; (iii) it cleaves concertedly both DNA strands at a single site during a single turnover on a three site plasmid to yield linear DNA. Cognate oligonucleotide added in trans increases the reaction velocity and changes the reaction pattern for the EcoRII cleavage of one and two-site plasmids but has little effect on the three-site plasmid. Taken together the data indicate that EcoRII requires simultaneous binding of three rather than two recognition sites in cis to achieve concerted DNA cleavage at a single site. We show that the orthodox type IIP enzyme PspGI which is an isoschisomer of EcoRII, cleaves different plasmid substrates with equal rates. Data provided here indicate that type IIE restriction enzymes EcoRII and NaeI follow different mechanisms. We propose that other type IIE restriction enzymes may employ the mechanism suggested here for EcoRII.     

EcoRV restriction endonuclease: communication between DNA recognition and catalysis.

A genetic system was constructed for the mutagenesis of the EcoRV restriction endonuclease and for the overproduction of mutant proteins. The system was used to make two mutants of EcoRV, with Ala in place of either Asn185 or Asn188. In the crystal structure of the EcoRV-DNA complex, both Asn185 and Asn188 contact the DNA within the EcoRV recognition sequence. But neither mutation affected the ability of the protein to bind to DNA. In the absence of metal ion cofactors, the mutants bound DNA with almost the same affinity as that of the wild-type enzyme. In the presence of Mg2+, both mutants retained the ability to cleave DNA specifically at the EcoRV recognition sequence, but their activities were severely depressed relative to that of the wild-type. In contrast, with Mn2+ as the cofactor, the mutant enzymes cleaved the EcoRV recognition site with activities that were close to that of the wild-type. When bound to DNA at the EcoRV recognition site, the mutant proteins bound Mn2+ ions readily, but they had much lower affinities for Mg2+ ions than the wild-type enzyme. This was the reason for their low activities with Mg2+ as the cofactor. The arrangement of the DNA recognition functions, at one location in the EcoRV restriction enzyme, are therefore responsible for organizing the catalytic functions at a separate location in the protein.     

VIRS: A visual tool for identifying restriction sites in multiple DNA sequences

VIRS (A visual tool for identifying restriction sites in multiple DNA sequences) is an interactive web‐based program designed for restriction endonuclease cut sites prediction and visualization. It can afford to analyze multiple DNA sequences simultaneously and produce visual restriction maps with several useful options intended for users' customization. These options also perform in‐depth analysis of the restriction maps, such as providing virtual electrophoretic result for digested fragments. Different from other analytical tools, VIRS not only displays visual outputs but also provides the detailed properties of restriction endonucleases that are commercially available. All the information of these enzymes is stored in our internal database, which is updated monthly from the manufacturers' web pages. It is freely available online at http://bis.zju.edu.cn/virs/index.html . © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Mapping of recognition sites for the restriction endonuclease from Escherichia coli K12 on bacteriophage PM2 DNA.

Several mutations in gene B of phage S13 appear to shorten the B protein by elimination of an N-terminal fragment, without destroying the B protein function. The shortened B protein resulting from each of these mutations can block the unique DNA-nicking properties of the S13 gene A protein. Because of the block in gene A function, normal gene B protein may have a function in phage DNA synthesis in addition to its known role in catalyzing capsid assembly.From gel electrophoresis the mutant B protein is estimated to be shorter than the normal S13 B protein by 1720 ± 70 daltons and is therefore believed to be an internal reinitiation fragment. The reinitiated fragments are functional and are made in about twice the amount of the normal B protein.The phage mutants which yield the reinitiation fragments are double mutants, each phage containing the same gene B nonsense mutation and each appearing to contain a different compensating gene B mutation. Various data support the assumption that the compensating mutations are frame-shifts, including the fact that suppression does not restore the normal-sized B protein. The reinitiation is assumed to occur at a pre-existing out-of-phase initiator codon, near the nonsense triplet; the correct reading frame would then be restored by each of the several different compensating mutations.The position of the normal S13 B protein in the gel electrophoresis pattern has been located both by elimination and shifting of the B peak, using appropriate amber mutants. The molecular weight of the S13 B protein is about 17,200, and is 2100 daltons less than the B protein of phage φX174; the S13 B protein can nevertheless substitute for the φX 174 B protein. Thus substantial portions of the B protein can be deleted without destroying its function.     

The specific non-symmetrical sequence recognized by restriction endonuclease MboII

The restriction endonuclease MboII, isolated from Moraxella bovis (ATCC 10900), cleaves bacteriophage φX174am3 replicative form I DNA into ten fragments. The physical map of these fragments has been aligned with the sequence of φX174 DNA. There is no sequence with 2-fold rotational symmetry common to the region of all ten cleavage sites. However, the non-symmetrical sequence 5′-G-A-A-G-A-3′ 3′-C-T-T-C-T-5′ occurs near to each cleavage site. Precise mapping of the cleavages in both DNA strands at several sites places the cuts eight nucleotides to the right of the upper sequence and seven nucleotides to the right of the lower sequence.     

A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity

A technique for conveniently radiolabeling DNA restriction endonuclease fragments to high specific activity is described. DNA fragments are purified from agarose gels directly by ethanol precipitation and are then denatured and labeled with the large fragment of DNA polymerase I, using random oligonucleotides as primers. Over 70% of the precursor triphosphate is routinely incorporated into complementary DNA, and specific activities of over 10(9) dpm/microgram of DNA can be obtained using relatively small amounts of precursor. These "oligolabeled" DNA fragments serve as efficient probes in filter hybridization experiments.     

DNA binding and recognition by the IIs restriction endonuclease MboII

The type IIs restriction endonuclease MboII recognizes nonsymmetrical GAAGA sites, cutting 8 (top strand) and 7 (bottom strand) bases to the right. Gel retardation showed that MboII bound specifically to GAAGA sequences, producing two distinct complexes each containing one MboII and one DNA molecule. Interference analysis indicated that the initial species formed, named complex 1, comprised an interaction between the enzyme and the GAAGA target. Complex 2 involved interaction of the protein with both the GAAGA and the cutting sites. Only in the presence of divalent metal ions such as Ca(2+) is the conversion of complex 1 to 2 rapid. Additionally, a very retarded complex was seen with Ca(2+), possibly a (MboII)(2)-(DNA)(2) complex. Plasmids containing a single GAAGA site were hydrolyzed slowly by MboII. Plasmids containing two sites were cut far more rapidly, suggesting that the enzyme requires two recognition sites in the same DNA molecule for efficient hydrolysis. MboII appears to have a mechanism similar to the best characterized type IIs enzyme, FokI. Both enzymes initially bind DNA as monomers, followed by dimerization to give an (enzyme)(2)-(DNA)(2) complex. Dimerization is efficient only when the two target sites are located in the same DNA molecule and requires divalent metal ions.     

RsrII--a novel restriction endonuclease with a heptanucleotide recognition site.

A sequence-specific endonuclease present in extracts of Rhodopseudomonas sphaeroides 630 has been purified and characterized. The enzyme, Rsr II, recognises and cleaves the palindromic heptanucleotide sequence: (sequence; see test) By virtue of its unusual specificity, RsrII cuts most DNA molecules very infrequently which should facilitate the physical mapping of large genomes.     

Effects on NaeI-DNA recognition of the leucine to lysine substitution that transforms restriction endonuclease NaeI to a topoisomerase: a model for restriction endonuclease evolution.

Substituting lysine for leucine at position 43 (L43K) transforms NaeI from restriction endonuclease to topoisomerase and makes NaeI hypersensitive to intercalative anticancer drugs. Here we investigated DNA recognition by Nael-L43K. Using DNA competition and gel retardation assays, NaeI-L43K showed reduced affinity for DNA substrate and the ability to bind both single- and double-stranded DNA with a definite preference for the former. Sedimentation studies showed that under native conditions NaeI-L43K, like NaeI, is a dimer. Introduction of mismatched bases into double-stranded DNA significantly increased that DNA's ability to inhibit NaeI-L43K. Wild-type NaeI showed no detectable binding of either single-stranded DNA or mismatched DNA over the concentration range studied. These results demonstrate that the L43K substitution caused a significant change in recognition specificity by NaeI and imply that NaeI-L43K's topoisomerase activity is related to its ability to bind single-stranded and distorted regions in DNA. A mechanism is proposed for the evolution of the NaeI restriction-modification system from a topoisomerase/ligase by a mutation that abolished religation activity and provided a needed change in DNA recognition.