The SalGI restriction endonuclease. Mechanism of DNA cleavage.

The cleavage of supercoiled DNA of plasmid pMB9 by restriction endonuclease SalGI has been studied. Under the optimal conditions for this reaction, the only product is the linear form of the DNA, in which both strands of the duplex have been cleaved at the SalGI recognition site. DNA molecules cleaved in one strand at this site were found to be poor substrates for the SalGI enzyme. Thus, both strands of the DNA appear to be cleaved in a concerted reaction. However, under other conditions, the enzyme cleaves either one or both strands of the DNA; the supercoiled substrate is then converted to either open-circle or linear forms, the two being produced simultaneously rather than consecutively. We propose a mechanism for the SalGI restriction endonuclease which accounts for the reactions of this enzyme under both optimal and other conditions. These reactions were unaffected by the tertiary structure of the DNA.     

A restriction endonuclease cleavage map of mouse mitochondrial DNA.

A restriction endonuclease cleavage map is presented for mouse mitochondrial DNA. This map was constructed by electron microscopic measurements on partial digests containing fixed D-loops, and by electrophoretic analysis of partial and complete single enzyme digests, and of double digests. No map differences were detected between mitochondrial DNA from cultured LA9 cells and an inbred mouse line for the six endonucleases used. Three cleavage sites recognized by HpaI, five sites recognized by HincII, two sites recognized PstI and four sites recognized by BamI were located with respect to the origin of replication and the EcoRI and HinIII sites previously determined by others. No cleavages were produced by KpnI or SalI. The migration of linear DNA with a molecular weight greater than 1 X 10(6) was not a linear function of log molecular weight in 1% agarose gels run at 6.6 volts/cm.     

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.     

The effect of sequence specific DNA methylation on restriction endonuclease cleavage.

Sequence specific DNA methylation sometimes results in the protection of some or all of a restriction endonucleases' cleavage sites. This is usually, but not always, the result of methylation of one or both strands of DNA at the site characteristic of the corresponding "cognate" modification methylase. The known effects of sequence specific methylation on restriction endonucleases are compiled.     

Site-dependent cleavage of pBR322 DNA by restriction endonuclease HinfI.

Cleavage of pBR322 DNA I by the restriction endonuclease HinfI is preferentially inhibited at specific HinfI cleavage sites. These sites in pBR322 DNA I have been identified and ordered with respect to the frequency with which they are cleaved. The HinfI site most resistant to cleavage in pBR322 DNA I is unique in that runs of G-C base pairs are immediately adjacent on both sites. Two differently permuted linear (DNA III) species were produced by cleavage with two different restriction endonucleases, PstI and AvaI. Only one of these linear molecules, that produced by PstI, exhibits the same preferential cleavage pattern as DNA I. The second linear species, that arising from AvaI digestion, shows pronounced relative inhibition of cleavage at the HinfI sites nearest the ends of the molecule (100 to 120 base pairs away, respectively). This result suggest that proximity to the termini of a linear DNA molecule might also influence preferential cleavage. The possibility of formation of stem-loop structures does not appear to influence preferential cleavage by HinfI.     

Altered restriction endonuclease cleavage pattern of simian virus 40 DNA.

Three different groups of temperature-sensitive mutants of simian virus 40, isolated and characterized by Chou and Martin (J. Virol. 13:1101--1109, 1974), have been analyzed by using restriction endonucleases. Differences between the restriction endonuclease cleavage pattern of these mutants and that of the standard simian virus 40 strain have been mapped. These include the following observations: (i) tsD202 carries a defective HaeIII cleavage site at position 0.9 map units; (ii) tsB204 exhibits a defective HaIII site at position 0.21 and a defective HinIII site at 0.655 map units, and (iii) tsC219 carries a new HinIII site at position 0.15. We have isolated a few wild-type revertants from each of the temperature-sensitive mutant strains; each displays the endonuclease cleavage pattern of its parental temperature-sensitive strain.     

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.     

Selective inhibition of restriction endonuclease cleavage by DNA intercalators

The preferred dye binding sites and the microenvironment of known nucleotide sequences within mitochondrial and plasmid pBR322 DNA was probed in a gross fashion with restriction endonucleases. The intercalating dyes, ethidium bromide and propidium iodide, do not inhibit a given restriction endonuclease equally at all of the restriction sites within a DNA molecule. The selective inhibition may be explained, in part, by the potential B to Z conformation transition of DNA flanking the restriction site and by preferred dye binding sites. Propidium iodide was found to be a more potent inhibitor than ethidium bromide and the inhibition is independent of the type of cut made by the enzyme.     

Modes of DNA cleavage by the EcoRV restriction endonuclease

The mechanism of action of the EcoRV restriction endonuclease at its single recognition site on the plasmid pAT153 was analyzed by kinetic methods. In reactions at pH 7.5, close to the optimum for this enzyme, both strands of the DNA were cut in a single concerted reaction: DNA cut in only one strand of the duplex was neither liberated from the enzyme during the catalytic turnover nor accumulated as a steady-state intermediate. In contrast, reactions at pH 6.0 involved the sequential cutting of the two strands of the DNA. Under these conditions, DNA cut in a single strand was an obligatory intermediate in the reaction pathway and a fraction of the nicked DNA dissociated from the enzyme during the turnover. The different reaction profiles are shown to be consistent with a single mechanism in which the kinetic activity of each subunit of the dimeric protein is governed by its affinity for Mg2+ ions. At pH 7.5, Mg2+ is bound to both subunits of the dimer for virtually the complete period of the catalytic turnover, while at pH 6.0 Mg2+ is bound transiently to one subunit at a time. The kinetics of the EcoRV nuclease were unaffected by DNA supercoiling.     

DNA recognition and cleavage by the EcoP15 restriction endonuclease.

EcoP15 is a restriction-modification enzyme coded by the P15 plasmid of Escherichia coli. We have determined the sites recognized by this enzyme on pBR322 and simian virus 40 DNA. The enzyme recognizes the sequence:

In restriction, the enzyme cleaves the DNA 25 to 26 base-pairs 3′ to this sequence to leave single-stranded 5′ protrusions two bases long.     

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.     

Polymorphism of mitochondrial DNA in pigs based on restriction endonuclease cleavage patterns

Restriction endonuclease cleavage patterns of mitochondrial DNA (mtDNA) of pigs and Japanese wild boars were analyzed using 17 enzymes which recognize six nucleotides. The map of cleavage sites was made by double-digestion methods. Polymophism of mtDNA was detected in the digestion by BglII, EcoRV, ScaI, and StuI. The restriction cleavage patterns were identical among the breeds of Landrace, Hampshire, Duroc I, and Large White I (A type). The patterns of Large White II were the same as those of Japanese wild boars (B type). A difference between the A type and the B type of mtDNA was found in the case of three restriction enzymes, BglII, ScaI, and StuI, and the nucleotide alterations between them were estimated as more than six. On the other hand, a difference between mtDNA from almost all pigs and mtDNA from Duroc II was detected using EcoRV. We suggest that the difference of mtDNA between the A type and the B type of mtDNA could result from the different origin of boars, that is, whether they were of European or Asian origin.     

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.     

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.     

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.     

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.     

Studies on the cleavage of bacteriophage lambda DNA with EcoRI restriction endonuclease

The five EcoRI² restriction sites in bacteriophage lambda DNA have been mapped at 0.445, 0.543, 0.656, 0.810, and 0.931 fractional lengths from the left end of the DNA molecule. These positions were determined electron-microscopically by single-site cleavage of hydrogen-bonded circular λ DNA molecules and by cleavage of various DNA heteroduplexes between λ DNA and DNA from well defined λ mutants. The DNA lengths of the EcoRI fragments are in agreement with their electrophoretic mobility on agarose gels but are not in agreement with their mobilities on polyacrylamide gels. These positions are different from those previously published by Allet et al. (1973). Partial cleavage of pure λ DNA by addition of small amounts of EcoRI endonuclease does not lead to random cleavage between molecules. Also, the first site cleaved is not randomly distributed among the five sites within a molecule. The site nearest the right end is cleaved first about ten times more frequently than either of the two center sites.     

Survey and mapping of restriction endonuclease cleavage sites in bacteriophage T7 DNA.

A survey of restriction endonucleases having different cleavage specificities has identified 10 that do not cut wild-type bacteriophage T7 DNA, 11 that cut at six or fewer sites, four that cut at 18 to 45 sites, and 12 that cut at more than 50 sites. All the cleavage sites for the 13 enzymes that cut at 26 or fewer sites have been mapped. Cleavage sites for each of the 10 enzymes that do not cut T7 DNA would be expected to occur an average of 9 to 10 times in a random nucleotide sequence the length of T7 DNA. A possible explanation for the lack of any cleavage sites for these enzymes might be that T7 encounters enzymes having these specificities in natural hosts, and that the sites have been eliminated from T7 DNA by natural selection. Five restriction endonucleases were found to cut within the terminal repetition of T7 DNA; one of these, KpnI, cuts at only three additional sites in the T7 DNA molecule. The length of the terminal repetition was estimated by two independent means to be approximately 155 to 160 base-pairs.     

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.     

Preferential site-dependent cleavage by restriction endonuclease PstI.

The four identical recognition sites for the restriction endonuclease PstI in purified plasmid pSM1 DNA I are cleaved at markedly different rates. The order and relative frequencies of cleavage at these four PstI sites have been determined from the order of appearance of partial cleavage products and from an analysis of production of specific unit length linear molecules. The same pattern of preferential cleavage is also found when linear, nicked circular, or relaxed closed circular forms of the same plasmid DNa are used as substrates for PstI. Inspection of the nucleotide sequences immediately adjoining each of the PstI sites suggests that the presence of adjacent runs of G-C base pairs confers significant resistance to cleavage.