Processive cleavage of concatemer DNA duplexes by Eco RII restriction endonuclease

Eco RII restriction endonuclease cleaves synthetic DNA-duplexes in which the recognition sites of this enzyme (5..CC _T ^A GG...) are repeated every 9 base pairs with the alternating orientation of the central AT pair. It operates in a processive mode, i.e. the bound enzyme molecule slides along the substrate toward neighboring recognition sites. Nona-nucleotides are the main products of the cleavage. The data obtained point to the capability of Eco RII endonuclease to recognize and cleave the substrate under both possible orientations of the central AT-pair of the recognition site with respect to the bound enzyme molecule. These data also show the close similarity of DNA structures in a complex with theenzyme and without.     

In vivo methylation by Escherichia coli K-12 mec+ deoxyribonucleic acid-cytosine methylase protects against in vitro cleavage by the RII restriction endonuclease (R. Eco RII).

We have analyzed the susceptibility of the deoxyribonucleic acid (DNA) of phage fd replicative form (RF) and of Escherichia coli to in vitro cleavage by purified RII restriction endonuclease (R. Eco RII). The results are summarized as follows: (i) fd, mec- RFI, isolated from infected E. coli K-12 mec- bacteria (a mutant strain lacking DNA-cytosine methylase activity), is cleaved into at least two fragments, whereas fd. mec+ RFI, isolated from the parental mec+ strain, is not cleaved. (ii) E. coli mec- DNA is extensively degraded, whereas mec+ DNA-cytosine methylase acts as an RII modification enzyme.     

Pentamidine-induced alteration in restriction endonuclease cleavage of plasmid DNA

We have used restriction enzymes and DNaseI as probes to determine the specificity of pentamidine binding to plasmid DNA. Cleavage of plasmid pAZ130 by EcoRI, EcoRV and ApaI is inhibited by pentamidine, cleavage by XbaI, NotI and AvaI is unaffected, while cleavage by XhoI, which recognizes the same sequence as AvaI, is stimulated. DNaseI footprinting of DNA containing these restriction sites revealed that pentamidine protection is not strictly limited to AT-rich regions. We suggest that perturbation of the DNA micro- environment by pentamidine binding is responsible for its effect on nucleases.     

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.     

Kinetic studies on the cleavage of adenovirus DNA by restriction endonuclease Eco RI.

The kinetics of cleavage of DNA from Adenovirus Type 1 (Ad1), Type 5 (Ad5) and Type 6 (Ad6) by restriction endonuclease EcoRI was investigated by quantitative evaluation of the fluorescence from ethidium stained DNA fragments separated on agarose gels. The apparent rate constants of cleavage at different cleavage sites have been determined and large differences in the cleavage rates of the individual sites within one type of DNA were found. From the kinetics of cleavage information on the sequence of the DNA fragments can be obtained. The order of the fragment A, B, C, D of Ad6 DNA obtained after complete cleavage by restriction endonuclease Eco RI was found to be A-D-C-B; the order of the corresponding fragments A, B, C of Ad1 and Ad5 DNA was found to be A-C-B.     

Effect of temperature and 4,6'-diamidine-2-phenylindole on restriction of supercoiled Col E1 DNA by Eco RI endonuclease.

Supercoiled Col E1 DNA is split by Eco RI endonuclease at 37 degrees C without intermediate formation of open circular DNA. Accumulation of this restriction product is observed at low temperature. The fluorescent dye, 4,6'-diamidine-2-phenylindole (DAPI) inhibits restriction by Eco RI endonuclease. This effect is due to the DAPI:DNA rather than to the DAPI:Eco RI interactions.     

Specific fragmentation of mitochondrial DNA from Neurospora crassa by restriction endonuclease Eco R I.

Linear, size-heterogenous mitochondrial DNA from $\text{Neurospora crassa}$ was cleaved by the restriction endonuclease Eco R I into eleven specific fragments. According to their contour lengths the fragments have molecular weights between 1.1 and 14 × 10⁶. The sum of the fragments lengths is identical with the contour length (19.8 μm, 41 × 10⁶ daltons) of the few circular molecules detectable in purified DNA preparations.The results suggest sequence homogeneity of mitochondrial DNA and further demonstrate that restriction enzymes can be used to establish a physical map of an unspecifically-fragmented DNA molecule.     

Transforming activity of plasmid and chromosomal DNA inEscherichia coli

An auxotrophic strain ofEscherichia coli with therecB recC sbcB genotype was transformed by chromosomal DNA of the prototrophic strain and by plasmid DNA carrying genes for antibiotic resistance (R1drd 19). The donor plasmid DNA obtained by cell lysis in the presence of Triton X-100 and subsequent centrifugation in a caesium chloride-ethidium bromide gradient was shown to have a circulaf molecule and to retain its completeness after penetration into the recipient. Experiments with mixtures or plasmid and chromosomal DNA indicate a competition between these two DNA types during the transformation reaction in the given system.     

Computer-assisted restriction endonuclease analysis of plasmid DNA in field strains of Salmonella enteritidis.

Computer-assisted restriction endonuclease analysis of plasmid DNA in field strains of Salmonella enterica serovar Enteritidis (S. enteritidis) is described. The procedure consists of plasmid DNA purification, its digestion with restriction endonuclease TaqI, electrophoresis, charge-coupled device camera scanning of the gels, and an analysis of the restriction patterns with the software Gel Manager. The system allowed us to analyse, in detail, results of plasmid profiling in more than 600 field strains of S. enteritidis. In addition to plasmid-free and virulence plasmid only containing strains, 15 additional plasmid types were detected. All the images and detailed protocols are available at the Web site http://www.clark.cz/vri/salmon.htm.     

Identification of gene products programmed by restriction endonuclease DNA fragments using an E. coli in vitro system.

DNA restriction enzyme fragments have been used to programme the synthesis of polypeptides in an in vitro system without apparent loss in fidelity compared with supercoiled templates. The system is extremely sensitive, less than 1 microgram of DNA can be used to direct the synthesis of 35S-labelled polypeptides of sufficiently high specific activity such that products can be identified by SDS-PAGE after a few hours autoradiography. The ability to analyse fragments can be used to readily assign specific proteins to small regions of the coding template, to identify cloned gene products distinct from those of the vector, and to identify cloned genes expressed from their own promoters. The in vitro system can be used successfully with bacterial DNA from other species and efficient extracts can be prepared from any E. coli K-12 strain, which should greatly facilitate the purification of factors controlling the expression of specific genes by complementation assay.     

Recombinational repair of chromosomal DNA double-strand breaks generated by a restriction endonuclease

DNA double-strand break repair can be accomplished by homologous recombination when a sister chromatid or a homologous chromosome is available. However, the study of sister chromatid double-strand break repair in prokaryotes is complicated by the difficulty in targeting a break to only one copy of two essentially identical DNA sequences. We have developed a system using the Escherichia coli chromosome and the restriction enzyme EcoKI, in which double-strand breaks can be introduced into only one sister chromatid. We have shown that the components of the RecBCD and RecFOR 'pathways' are required for the recombinational repair of these breaks. Furthermore, we have shown a requirement for SbcCD, the prokaryotic homologue of Rad50/Mre11. This is the first demonstration that, like Rad50/Mre11, SbcCD is required for recombination in a wild-type cell. Our work suggests that the SbcCD-Rad50/Mre11 family of proteins, which have two globular domains separated by a long coiled-coil linker, is specifically required for the co-ordination of double-strand break repair reactions in which two DNA ends are required to recombine at one target site.