Differential inhibition of a restriction enzyme by quinoxaline antibiotics

The inhibition of cleavage by HpaI at two well-defined restriction sites in linearised phi X174-RF DNA by quinoxaline antibiotics has been investigated. Echinomycin, which displays a certain preference for binding to GC basepairs, inhibits cleavage at one site much more than the other, whereas triostin A, which displays less pronounced sequence-selectivity, inhibits both sites about equally. Other congeners inhibit reaction at the two sites with varying effectiveness. The results demonstrate the usefulness of studying inhibition of cleavage at specific sites by restriction enzymes as a means of exploring the specificity of DNA-ligand interactions.     

Heparin inhibits EcoRI endonuclease cleavage of DNA at certain EcoRI sites.

Studies presented here demonstrate that heparin inhibits EcoRI endonuclease cleavage of DNA whereas related proteoglycans show no effect. The inhibition occurs at particular EcoRI sites that are near or overlap with palindromic sequences in the murine lambda 5 and Lyt-2 genes. Endogenous heparin from peritoneal mast cells co-isolates with DNA and inhibits digestion of peritoneal cell DNA at the inhibitable sites. Digestion of spleen DNA is inhibited at the same sites when commercial heparin is added prior to digestion. In both cases, the inhibition is abolished by pre-treating the DNA with heparinase. Thus, potential artifacts in restriction fragment length analyses could occur with DNA isolated either from cells that are naturally rich in heparin or from cells to which heparin has been added, e.g., as an anticoagulant.     

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.     

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.     

The inhibition of restriction endonuclease PvuII cleavage activity by methylation outside its recognition sequence.

The recombinant plasmid pGEM4Z-ras DNA which was methylated on dam and dcm sites outside the PvuII recognition sequence was digested with restriction endonuclease PvuII, and one of the three PvuII sites was about 16-fold less efficient to cleave than either of the other two. On the contrary, the three PvuII sites were cleaved at about the same rate on the unmethylated DNA molecule. The results show that the cleavage inhibition of the methylated DNA on the certain PvuII site was caused by methylation outside the PvuII recognition sequence. Maybe a adjacent methylated dam site *A was responsible for the less efficient cleavage. This observation suggests that methylation outside the recognition sequence may be considered a new factor in the kinetic experiment of restriction endonuclease.     

Sequence specific inhibition of DNA restriction enzyme cleavage by PNA.

Plasmids containing double-stranded 10-mer PNA (peptide nucleic acid chimera) targets proximally flanked by two restriction enzyme sites were challenged with the complementary PNA or PNAs having one or two mismatches, and the effect on the restriction enzyme cleavage of the flanking sites was assayed. The following PNAs were used: T10-LysNH2, T5CT4-LysNH2 and T2CT2CT4-LysNH2 and the corresponding targets cloned into pUC 19 were flanked by BamH1, Sal1 or Pstl sites, respectively. In all cases it was found that complete inhibition of restriction enzyme cleavage was obtained with the complementary PNA, a significantly reduced effect was seen with a PNA having one mismatch, and no effect was seen with a PNA having two mismatches. These results show that PNA can be used as sequence specific blockers of DNA recognizing proteins.     

A physical map of belomycin-specific fragmentation sites on PM2 bacteriophage DNA

Bleomycin treatment of PM2 DNA results in fragmentation of the genome at several specific sites. Application of restriction endonuclease digestion followed by bleomycin treatment has provided the basis for constructing a physical map of bleomycin fragmentation sites. Eleven sites have been located on the physical map relative toHpa II,Pst I, andHindIII cleavage sites. The fragmentation sites are not clustered in a particular region of the PM2 genome but 3 of the 11 sites do occur between theHpa II andPst I cleavage sites, a segment of DNA which comprises 14% of the PM2 DNA length.     

Mitochondrial DNA polymorphism in native Philippine cattle based on restriction endonuclease cleavage patterns

An analysis of patterns of cleavage of mtDNA by restriction endonucleases was performed for nine individuals from the Philippine population of native cattle. MtDNA polymorphisms were detected in the restriction patterns generated by the following six enzymes,BamHI,BglII,EcoRV,HindIII,PstI, andScaI. The restriction patterns showing polymorphisms were distributed nonrandomly among the nine individuals examined from the Philippine population of native cattle, indicating the existence of two separate types of mtDNA. These two types of mtDNA are very different from each other, at the level of subspecies. Since the native Philippine cattle are considered to represent an admixture of European and Indian cattle, the two types of mtDNA must be derived from the mtDNAs of both varieties. The polymorphic sites in mtDNA have been located on a restriction map, and the nucleotide substitutions at some of the sites have also been estimated.     

DNA restriction dependent on two recognition sites: activities of the SfiI restriction-modification system in Escherichia coli

In contrast to many type II restriction enzymes, dimeric proteins that cleave DNA at individual recognition sites 4-6 bp long, the SfiI endonuclease is a tetrameric protein that binds to two copies of an elongated sequence before cutting the DNA at both sites. The mode of action of the SfiI endonuclease thus seems more appropriate for DNA rearrangements than for restriction. To elucidate its biological function, strains of Escherichia coli expressing the SfiI restriction-modification system were transformed with plasmids carrying SfiI sites. The SfiI system often failed to restrict the survival of a plasmid with one SfiI site, but plasmids with two or more sites were restricted efficiently. Plasmids containing methylated SfI sites were not restricted. No rearrangements of the plasmids carrying SfiI sites were detected among the transformants. Hence, provided the target DNA contains at least two recognition sites, SfiI displays all of the hallmarks of a restriction-modification system as opposed to a recombination system in E. coli cells. The properties of the system in vivo match those of the enzyme in vitro. For both restriction in vivo and DNA cleavage in vitro, SfiI operates best with two recognition sites on the same DNA.     

Differential reactivities at restriction enzyme sites

A method has been developed to measure the rates of digestion by restriction enzymes at individual sites. This involves a simple arithmetical treatment of the integrated areas from a densitometer scan of an ethidium bromide stained gel. We have used this method to study the digestion by HpaI, HincII and SalI of pBR322 and phi X174 DNA, and the effect of various DNA binding ligands. One of the two HpaI sites in phi X174 DNA is much more sensitive to inhibition by ligands such as netropsin, which display a preference for AT base pairs, than is the other site. Inspection of the sequences flanking the restriction sites shows that the former contains a much higher proportion of AT base-pairs than dose the latter. The opposite phenomenon is observed with the two HincII sites in pBR322. This illustrates the importance of neighbouring sequences in the interaction between restriction enzymes and their cleavage sites in DNA.     

Studies of Viable T4 Bacteriophage Containing Cytosine-substituted DNA (T4dC Phage). III. Advantage of T4dC DNA as Substrate for Assay of Restriction Endonucleases

Cytosine-substituted phage T4 DNA (T4dC DNA) was demonstrated to be a splendid substrate for the assay of restriction endonucleases by agarose gel electrophoresis. For preparing those which cleave lambda phage DNA at few sites, T4dC DNA having appreciable number of cleavage sites was especially useful. As typical examples SalI and XbaI restriction endonucleases were chosen and an advantage of T4dC DNA for the enzyme unit determination was described. Screening of new restriction endonucleases from Streptomyces strains was facilitated by using T4dC DNA as a substrate for the assay.     

Versatile method employing basic techniques of genetic engineering to study the ability of low-molecular-weight compounds to bind covalently with DNA in cell-free systems

Numerous antitumor and carcinogenic compounds and free radicals are able to modify DNA by forming covalent bonds, mainly with nucleophilic centers in nucleobases. Such a binding is usually of utmost importance for the biological outcome. The level of DNA adducts formed by a given agent is in most cases extremely low; hence their detection is very difficult. Here we propose a simple approach, exploiting techniques widely used in genetic engineering, to demonstrate and characterize the covalent modification of a DNA fragment by any low-molecular-weight compound of interest in a cell-free system. The specifically designed, several-hundred-base-pairs-long double-stranded deoxyoligonucleotide (PCR amplified)--subject to modification--includes two restriction sites: one containing only GC base pairs recognized by restriction endonuclease MspI and the other including only AT base pairs recognized by restriction endonuclease Tru1I. The covalent modification of the restriction sites abolishes their recognition and thus cleavage by the endonucleases applied. The formation of DNA adducts is induced by incubating the oligonucleotide with increasing concentrations of a studied compound, in the appropriate activating system if required. Then, the modified oligonucleotide is submitted to digestion by the above-mentioned restriction endonucleases and the DNA fragments are separated by polyacrylamide gel electrophoresis. The inhibition of cleavage indicates the occurrence of covalent modification of the restriction site(s) while simultaneously pointing at the kind of base pairs involved in DNA adduct formation. The validation of the method was performed for two DNA binding antitumor compounds, cisplatin and CC-1065, which form adducts preferentially with guanine and adenine, respectively.     

Physical mapping of bacteriophage T4

Summary The 134 positions of the cleavage sites of the restriction endonucleases XbaI, HaeII and EcoRI were determined for a cytosine-containing DNA of bacteriophage T4. This physical map was aligned with the genetic map. The T4 early regions were further identified by hybridization of RNA synthesized in vitro to the restriction fragments and two promoter regions were localized by filter binding tests and R-loop analysis.     

Mapping of additional restriction enzyme cleavage sites on bacteriophage MB78 genome

A physical map of bacteriophage MB78 DNA indicating the cleavage sites for the enzymeBglII,ClaI,EcoRI,PvuII,SalI andSmaI comprising of a total of 34 cleavage sites have been constructed earlier. The cleavage sites for a few more restriction endonucleases likeApaI,AvaI,BglI,HindIII,KpnI andXhoI have now been mapped. A total of 72 cleavage sites on MB78 DNA are known by now. Relative positions ofEcoRI I and J fragments which could not be decided earlier has now been determined.     

Pig mitochondrial DNA: Polymorphism,restriction map orientation,and sequence data

Restriction endonuclease cleavage patterns of mitochondrial DNA (mtDNA) in pigs were analyzed using 18 enzymes which recognize six nucleotides and 1 four-nucleotide-recognizing enzyme. Pigs including Taiwan native breeds and miniature strains maintained in Japan were examined in this study; four commercial breeds of pigs and Japanese wild boars have been investigated earlier [Watanabe, T., et al. (1985). Biochem. Genet. 23:105]. mtDNA polymorphisms were observed in the cleavage patterns of five restriction enzymes, Bg1II, EcoRV, ScaI, StuI, and TaqI. The results support the previous hypothesis that pigs must be derived from two different maternal origins, European and Asian wild boars, and that a breed, Large White, arises from both European and Asian pigs. Two HindIII cleavage fragments were cloned into the HindIII site of M13mp10 and were partially sequenced by the dideoxynucleotide-chain termination method. Furthermore, DraI and StuI cleavage sites were newly determined on the restriction endonuclease map. On the basis of these results, the restriction endonuclease cleavage map of pig mtDNA was rewritten. Comparing sequence data of pig mtDNA at 237 positions with those of cow, human, mouse, and rat mtDNA, the sequence difference, silent and replacement changes, and transitions and transversions among mammalian species were estimated. The relationships among them are discussed.     

Segments of mitochondrial DNA of yeast carrying antibiotic resistances

Summary Mitochondrial DNA was isolated from an oligomycin-resistant petite mutant of yeast, Saccharomyces cerevisiae. It had repeated sequences of 3600 base pairs. This segment was about one twentieth of the whole mtDNA of wild type yeast, which had a size of 74 kilo base pairs.This segment of mtDNA had one cleavage site for a restriction endonuclease, Hind II, which was more resistant to cleavage than the other Hind II sites in wild type mtDNA. It had two cleavage sites for Hha I and gave two Hha fragments, which were arranged alternatively. Digestion with Hae III gave four fragments and these fragments were mapped.Mitochondrial DNA of this mutant showed a loss of heterogeneity in a melting profile. It melted within a narrow range of temperature, which was similar to that of poly dA·poly dT. Its differential melting curve was significantly different from that of wild type mtDNA.Mapping of mtDNA of a wild type yeast was carried out with restriction endonucleases. Fragments of mtDNA, which were isolated from petites carrying oligomycin-erythromycin-chloramphenicol-resistance and erythromycin-chloramphenicol resistance were also mapped. Loci of oligomycin-resistance, erythromycin-resistance and chloramphenicol-resistance were investigated based on the maps of Eco R I fragments and Hind II fragments.     

New procedure using a psoralen derivative for analysis of nucleosome associated DNA sequences in chromatin of living cells.

Sites for restriction endonuclease cleavage in double helical DNA are blocked from cleavage when the photoaffinity drug trimethylpsoralen is photobound at or near the site. In general, Hind III sites are about 15 fold more sensitive to inactivation than the other restriction sites which were tested, although sensitivity of different Hind III sites seems to vary somewhat depending on base sequences adjacent to the site. Hind III sites can be inactivated in two ways; one which completely blocks action of the specific restriction endonuclease and one permitting the introduction of a swivel which relaxes DNA supercoiling without producing a double strand break. Nucleosomes and perhaps other protein-DNA complexes can protect the underlying DNA sequence from trimethylpsoralen photobinding and thus protect restriction sites from inactivation. This property can be exploited to determine if specific sites are accessible to the psoralon probe in vivo and thus to establish if specific nucleotide sequences are nucleosome associated. Using this procedure evidence is obtained that nucleosomes on SV40 DNA in living infected cells are either distributed randomly or at many discrete alternate sites that approach a random distribution.     

EcoRII can be activated to cleave refractory DNA recognition sites.

EcoRII restriction sites [5'-CC(A/T)GG] in phage T3 and T7 DNA are refractory to cleavage by EcoRII, but become sensitive to cleavage in the presence of DNAs which contain an abundance of EcoRII sensitive sites (e.g. pBR322 or lambda DNA). Studies using fragments of pBR322 containing different numbers of EcoRII sites show that the susceptibility to EcoRII cleavage is proportional to the number of sites in the individual fragment. We postulate that EcoRII is the prototype of restriction endonucleases which require at least 2 simultaneously bound substrate sites for their activation. EcoRII sites are refractory when they occur at relatively low frequency in the DNA. The restriction enzyme can be activated by DNA with a higher frequency of sites.