A restriction endonuclease cleavage map of bacteriophage P2

Summary A restriction endonuclease cleavage map of phage P2 was constructed. The enzymes used and, within parenthesis, the number of their cleavage sites on the P2 lg cc DNA molecule were: AvaI(3), BalI(1), BamI(3), BglII(3), HaeIII (more than 40; only three were mapped), HindIII(0), HpaI(10), KpnI(3), PstI(3), SalI(2) and SmaI(1). The EcoRI cleavage sites (3), as determined earlier, were used as reference points for this study. The DNAs of a variety of P2 mutants carrying chromosomal aberrations (del1, del2, del3, del6, vir22, vir37(2), vir79 and vir94) were also similarly examined.     

The restriction endonuclease cleavage map of rat liver mitochondrial DNA.

Mitochondrial DNA from rat liver contains six sites for cleavage by the restriction endonucleases Hind III and EcoRI. A large stretch of DNA, comprising about 40% of the mitochondrial genome is not cleaved by either of the enzymes; eight cleavage sites are located on a DNA stretch of 35% of the genome length suggestive of an unequal distribution of the A - T baspairs over the molecule. The number of Hind III and Eco R I fragments is much higher than reported for other mammalian mitochondrial DNAs up to now.     

EcoRI restriction endonuclease cleavage site map of bacteriophage P22DNA.

The F plasmid is able to co-transfer (mobilize) the small, chimeric R plasmid pBR322 during conjugation only at a very low frequency (Bolivar et al., 1977). Mobilization has been found here to be invariably (> 99%) associated with a structural alteration of pBR322. The alteration was shown, by restriction endonuclease analysis and electron microscopy, to be an insertion of the F attachment sequence λδ (2.8 to 8.5F). λδ is, therefore, an insertion sequence.     

A restriction endonuclease cleavage map of mitochondrial DNA from transformed hamster cells.

Mitochondrial DNA from cultured C13/B4 hamster cells was cleaved by the restriction endonucleases Hpa II, Hind III, Eco RI and Bam HI into 7, 5, 3 and 2 unique fragments, respectively. The summed molecular weights of fragments obtained from electrophoretic mobilities in agarose-ethidium bromide gels (with Hpa I-cleaved T7 DNA as standard) and electron microscopic analysis of fragment classes isolated from gels (with SV40 DNA as standard) were in good agreement with the size of 10.37 +/- 0.22 x 10(6) daltons (15,700 +/- 330 nucleotide pairs) determined for the intact circular mitochondrial genome. Cyclization of all Hind III, Eco RI and Bam HI fragments was observed. A cleavage map containing the 17 restriction sites (+/- 1% s.d.) was constructed by electrophoretic analysis of 32P-labeled single- and double-enzyme digestion products and reciprocal redigestion of isolated fragments. The 7 Hpa II sites were located in one half of the genome. The total distribution of the 17 cleavages around the genome was relatively uniform. The position of the D-loop was determined from its location and expansion on 3 overlapping restriction fragments.     

Cleavage of phosphorothioated DNA and methylated DNA by the type IV restriction endonuclease ScoMcrA

Many taxonomically diverse prokaryotes enzymatically modify their DNA by replacing a non-bridging oxygen with a sulfur atom at specific sequences. The biological implications of this DNA S-modification (phosphorothioation) were unknown. We observed that simultaneous expression of the dndA-E gene cluster from Streptomyces lividans 66, which is responsible for the DNA S-modification, and the putative Streptomyces coelicolor A(3)2 Type IV methyl-dependent restriction endonuclease ScoA3McrA (Sco4631) leads to cell death in the same host. A His-tagged derivative of ScoA3McrA cleaved S-modified DNA and also Dcm-methylated DNA in vitro near the respective modification sites. Double-strand cleavage occurred 16-28 nucleotides away from the phosphorothioate links. DNase I footprinting demonstrated binding of ScoA3McrA to the Dcm methylation site, but no clear binding could be detected at the S-modified site under cleavage conditions. This is the first report of in vitro endonuclease activity of a McrA homologue and also the first demonstration of an enzyme that specifically cleaves S-modified DNA.     

Rational engineering of type II restriction endonuclease DNA binding and cleavage specificity

The type II restriction endonucleases are indispensible tools for molecular biology. Although enzymes recognizing nearly 300 unique sequences are known, the ability to engineer enzymes to recognize any sequence of choice would be valuable. However, previous attempts to engineer new recognition specificity have met limited success. Here we report the rational engineering of multiple new type II specificities. We recently identified a family of MmeI-like type II endonucleases that have highly similar protein sequences but different recognition specificity. We identified the amino-acid positions within these enzymes that determine position specific DNA base recognition at three positions within their recognition sequences through correlations between their aligned amino-acid residues and aligned recognition sequences. We then altered the amino acids at the identified positions to those correlated with recognition of a desired new base to create enzymes that recognize and cut at predictable new DNA sequences. The enzymes so altered have similar levels of endonuclease activity compared to the wild-type enzymes. Using simple and predictable mutagenesis in this family it is now possible to create hundreds of unique new type II restriction endonuclease specificities. The findings suggest a simple mechanism for the evolution of new DNA specificity in Nature.     

Identification of a new strain of adenovirus type 2 by restriction endonuclease analysis

Restriction endonuclease analysis has been used to identify a new strain Ad2a of human adenovirus type 2 (Ad2). The sites for six restriction endonucleases have been mapped on the Ad2a genome and compared with those for the Ad2 prototype.     

Structure of bovine papillomavirus type 1 DNA in a transformed mouse cell line

Linearized bovine papillomavirus type 1 (BPV-1) DNA was introduced into mouse C127 cells, where it recircularized and replicated as an intact monomeric, extrachromosomal circular form in the resulting transformants. These cells contained a mixture of complex high molecular weight forms that were converted to a linear form of approximately BPV-1 size upon digestion with an enzyme that cuts once within the BPV-1 genome. Further analysis of one of these cell lines revealed that these high molecular weight forms consisted of two components. One was detected on agarose gels as a diffuse smear of slow-migrating material representing linear forms that were tightly associated with host chromosomes, probably by integration. The second component was composed of discrete-sized oligomeric open and supercoiled extrachromosomal circular forms of up to approximately 48 X 10(3) base-pairs (6 tandemly linked BPV-1 genomes) in size. No catenated (interlocked) forms could be detected.     

BcefI, a new type IIS restriction endonuclease.

A new Type IIS restriction endonuclease was identified, partially purified and characterized from a Bacillus cereus subsp. fluorescens strain. The enzyme recognizes the nonpalindromic sequence ACGGC and cleaves at a distance from it. The cleavage appears to occur with a +/- 1 basepair uncertainty. Thus the cleavage and recognition site is as shown below: ACGGC(N)11-13 TGCCG(N)12-14.     

Identification of unintegrated forms of Kirsten murine sarcoma viral DNA and restriction endonuclease cleavage map of linear DNA.

We detected unintegrated linear 7.0-kilobase pair DNA and covalently closed circular DNA species in NIH3T3 cells recently infected with Kirsten murine sarcoma virus. Using the linear DNA, we constructed a restriction endonuclease cleavage map and compared it with the map of Harvey murine sarcoma virus. The restriction endonuclease maps of two segments, one 1.2 kilobase pairs (SmaI site) to 3.7 kilobase pairs (HindIII site) from the right end (corresponding to the viral 3' side) and the other 0.5 kilobase pair (SmaI and KpnI sites) to 0.9 kilobase pair (KpnI site) from the left end, were identical in the two virus types.     

Stimulation of cellular DNA synthesis by wild type and mutant bovine papillomavirus DNA

Microinjection of recombinant plasmids containing bovine papillomavirus type 1 DNA into the nuclei of mouse C127 cells results in the stimulation of cellular DNA synthesis. Mutations in the viral E2 gene have no apparent effect on this activity even though the same mutations prevent efficient C127 cell focus formation and inhibit transactivation by this gene.     

Subunit assembly for DNA cleavage by restriction endonuclease SgrAI

The SgrAI endonuclease usually cleaves DNA with two recognition sites more rapidly than DNA with one site, often converting the former directly to the products cut at both sites. In this respect, SgrAI acts like the tetrameric restriction enzymes that bind two copies of their target sites before cleaving both sites concertedly. However, by analytical ultracentrifugation, SgrAI is a dimer in solution though it aggregates to high molecular mass species when bound to its specific DNA sequence. Its reaction kinetics indicate that it uses different mechanisms to cleave DNA with one and with two SgrAI sites. It cleaves the one-site DNA in the style of a dimeric restriction enzyme acting at an individual site, mediating neither interactions in trans, as seen with the tetrameric enzymes, nor subunit associations, as seen with the monomeric enzymes. In contrast, its optimal reaction on DNA with two sites involves an association of protein subunits: two dimers bound to sites in cis may associate to form a tetramer that has enhanced activity, which then cleaves both sites concurrently. The mode of action of SgrAI differs from all restriction enzymes characterised previously, so this study extends the range of mechanisms known for restriction endonucleases.     

Physical maps of bovine papillomavirus type 1 and type 2 genomes.

Physical maps of bovine papillomavirus type 1 and type 2 (BPV-1 and BPV-2) DNA were constructed from analysis of the electrophoretic mobilities of restriction endonuclease cleavage fragments from dual digests. BPV-1 DNA was sensitive to Hind III, HindIII, EcoRI, HpaI, AND BamHI, with all but HindII yielding single scissions. BPV-2 DNA was resistant to EcoRI, and HindIII had one cleavage site whereas HpaI, BamHI, and HindII yielded multiple fragments. Of four BPV-1 isolates examined, DNA from one isolate was resistant to HindIII, and another DNA isolate was resistant to BamHI. The three BPV-2 isolates examined were uniformly sensitive to the restriction endonucleases employed.     

A detailed restriction endonuclease site map of theZea mays plastid genome

Fragments produced by partial digestion of plastid DNA fromZea mays withEco RI were cloned in Charon 4A. A circular, fine structure physical map of the plastid DNA was then constructed from restriction endonucleaseSal I,Pst I,Eco RI, andBam HI recognition site maps of cloned overlapping segments of the plastid genome. These fragments were assigned molecular weights by reference to size markers from both pBR322 and lambda phage DNA. Because of the detail and extent of the derived map, it has been possible to construct a coordinate system which has a unique zero point and within which all the restriction fragments and previously described structural features can be mapped. A computer program was constructed which will display in a circular fashion any of the above features using an X-Y plotter.     

Crystallization of restriction endonuclease BamHI with nonspecific DNA

Restriction endonucleases show extraordinary specificity in distinguishing specific from nonspecific DNA sequences. A single basepair change within the recognition sequence results in over a million-fold loss in activity. To understand the basis of this sequence discrimination, it is just as important to study the nonspecific complex as the specific complex. We describe here the crystallization of restriction endonuclease BamHI with several nonspecific oligonucleotides. The 11-mer, 5'-ATGAATCCATA-3', yielded cocrystals with BamHI, in the presence of low salt, that diffracted to 1.9 A with synchrotron radiation. The cocrystals belong to the space group P2(1)2(1)2(1) with unit cell dimensions of a = 114.8 A, b = 91.1 A, c = 66.4 A, alpha = 90 degrees, beta = 90 degrees, gamma = 90 degrees. This success in the cocrystallization of BamHI with a nonspecific DNA provides insights for future attempts at crystallization of other nonspecific DNA-protein complexes.     

Isolation and restriction endonuclease analysis of mycobacterial DNA

A method for the isolation of DNA from mycobacteria propagated in vitro is described that utilizes organic solvents to extract lipoidal components from the outer membrane, and digestion with a protease (nagarse) and lysozyme to penetrate the cell wall. The mycobacterial cells were lysed by the addition of detergent and the DNA was purified by digestion with pronase, sequential phenol and chloroform extractions, and digestion with RNAase A. The isolated DNA, which was obtained in good yields, was of a relatively high Mr and could be readily digested by restriction endonucleases. By this method, the genomes of Mycobacterium avium, M. intracellulare, M. lepraemurium, 'M. lufu', M. marinum, M. phlei, M. scrofulaceum, M. smegmatis and M. tuberculosis were isolated and the restriction endonuclease digestion patterns analysed. Each species could be distinguished by the digestion patterns, indicating that this approach can be used for identifying mycobacterial species. This approach is also sufficiently sensitive to differentiate strains since we were able to distinguish two independently isolated strains of M. tuberculosis, H37 and H4. In addition, no evidence was obtained for the presence of methylcytosine residues in the sequences 5'.CCGG.3',5'.CCCGGG.3',5'.CC(A/T) GG.3' or for methyladenine at 5'.GATC.3' in the DNA of the nine mycobacterial species examined using pairs of restriction enzymes that recognize and cleave at the same nucleotide sequence but differ in their sensitivity to 5-methylcytosine or 6N-methyladenine.     

Multivariate analysis of Neisseria DNA restriction endonuclease patterns

Chromosomal DNA was extracted from eleven Neisseria meningitidis and seven Neisseria gonorrhoeae isolates and cleaved with the restriction enzyme HindIII. The DNA fragments were separated according to their size, using a 4% polyacrylamide gel. The band patterns obtained were digitized and statistically analysed by the SIMCA method. To develop the models for N. meningitidis (class 1) and N. gonorrhoeae (class 2), all eleven meningococci and seven gonococci, were used. All strains were classified correctly and showed an extremely good class separation.     

Efficient manipulation of nanoparticle-bound DNA via restriction endonuclease

As a programmable biopolymer, DNA has shown great potential in the fabrication and construction of nanometer-scale assemblies and devices. In this report, we described a strategy for efficient manipulation of gold nanoparticle-bound DNA using restriction endonuclease. The digestion efficiency of this restriction enzyme was studied by varying the surface coverage of stabilizer, the size of nanoparticles, as well as the distance between the nanoparticle surface and the enzyme-cutting site of particle-bound DNA. We found that the surface coverage of stabilizer is crucial for achieving high digestion efficiency. In addition, this stabilizer surface coverage can be tailored by varying the ion strength of the system. Based on the results of polyacrylamide gel electrophoresis and fluorescent study, a high digestion efficiency of 90+% for particle-bound DNA was achieved for the first time. This restriction enzyme manipulation can be considered as an additional level of control of the particle-bound DNA and is expected to be applied to manipulate more complicated nanostructures assembled by DNA.