An electron microscopic method for studying and mapping the region of weak sequence homology between simian virus 40 and polyoma DNAs.

A procedure for investigating the possibility of small amounts of partial DNA sequence homology between two defined DNA molecules has been developed and used to test for sequence homology between simian virus 40 and polyoma DNAs. This procedure, which does not necessitate the use of separated viral DNA strands, involves the construction of hybrid DNA molecules containing a simian virus 40 DNA molecule covalently joined to a polyoma DNA molecule, using the sequential action of EcoRI restriction endonuclease and Escherichia coli DNA ligase. Denaturation of such hybrid DNA molecules then makes it possible to examine intramolecularly rather than intermolecularly renatured molecules. Visualization of these intramolecularly renatured “snapback” molecules with duplex regions of homology by electron microscopy reveals a 15% region of weak sequence homology. This region is denatured at about 35 °C below the melting temperature of simian virus 40 DNA and therefore corresponds to about 75% homology. This region was mapped on both the simian virus 40 and polyoma genomes by the use of Hemophilus parainfluenzae II restriction endonuclease cleavage of the simian virus 40 DNA prior to EcoRI cleavage and construction of the hybrid molecule. The 15% region of weak homology maps immediately to the left of the EcoRI restriction endonuclease cleavage site in the simian virus 40 genome and halfway around from the EcoRI restriction endonuclease cleavage site in the polyoma genome.     

Action of Escherichia coli P1 restriction endonuclease on simian virus 40 DNA

The P1 restriction endonuclease (EcoP1) prepared from a P1 lysogen of Escherichia coli makes one double-strand break in simian virus (SV40) DNA. In the presence of cofactors S-adenosylmethionine and ATP the enzyme cleaves 70% of the closed circular SV40 DNA molecules once to produce unit-length linear molecules and renders the remaining 30% resistant to further cleavage. No molecules were found by electron microscopy or by gel electrophoresis that were cleaved more than once. It would appear that the double-strand break is made by two nearly simultaneous single-strand breaks, since no circular DNA molecules containing one single-strand break were found as intermediates during the cleavage reaction. The EcoP1 endonuclease-cleaved linear SV40 DNA molecules are not cleaved at a unique site, as shown by the generation of about 65% circular molecules after denaturation and renaturation. These EcoP1 endonuclease-cleaved, renatured circular molecules are resistant to further cleavage by EcoP1 endonuclease.The EcoP1 endonuclease cleavage sites on SV40 DNA were mapped relative to the partial denaturation map and to the EcoRI and HpaII restriction endonuclease cleavage sites. These maps suggest there are a minimum of four unique but widely spaced cleavage sites at 0.09, 0.19, 0.52, and 0.66 SV40 units relative to the EcoRI site. The frequency of cleavage at any particular site differs from that at another site. If S-adenosylmethionine is omitted from the enzyme reaction mix, SV40 DNA is cleaved into several fragments.An average of 4.6 ± 1 methyl groups are transferred to SV40 DNA from S-adenosylmethionine during the course of a normal reaction containing the cofactors. Under conditions which optimize this methylation, 7 ± 1 methyl groups can be transferred to DNA. This methylation protects most of the molecules from further cleavage. The methyl groups were mapped relative to the Hemophilus influenzae restriction endonuclease fragments. The A fragment receives three to four methyl groups and the B and G fragments each receive one to two methyl groups. These fragments correspond to those in which cleavage sites are located.     

Triplication of a unique genetic segment in a simian virus 40-like virus of human origin and evolution of new viral genomes

The non-defective (heavy) virions from a simian virus 40-like virus (DAR virus) isolated from human brain have been serially passaged at high input multi-plicities in primary monkey kidney cells. The ³²P-labeled, progeny DAR-viral genomes have been purified and tested for sensitivity to the R_I restriction endouclease from Escherichia coli (Eco RI3 restriction nuclease). The parental DAR-viral genomes share many physical properties with “standard” simian virus 40 DNA and are cleaved once by the Eco RI restriction nuclease. After the fourth serial passage, three populations of genomes could be distinguished: Eco RI resistant, Eco RI sensitive (one cleavage site) and Eco RI “supersensitive” (three, symmetrically-located, cleavage sites). The Eco RI cleavage product of the “supersensitive” form is one-third the physical size (10.4 S) of simian virus 40 DNA and reassociates about three times more rapidly than sheared, denatured simian virus 40 DNA. From the fourth to the eighth serial passages, the genomes containing this specific triplication of viral DNA sequences were selected for and became the predominant viral DNA species.     

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.     

The DNA sequence on bacteriophage G4 recognized by the Escherichia coli B restriction enzyme.

Bacteriophage G4 possesses a single EcoB site located in the overlap between restriction fragments HinfI-12 and HaeIII-6. The sequence 5′-T-G-A … 8N … T-G-C-T occurs once in this segment and nowhere else in the DNA sequence of G4. Four independent G4 mutants that were not restricted by Escherichia coli B possessed the sequence 5′-T-G-A … 8N … T-G-C-C. The common sequence shared by the previously mapped EcoB sites on φXsB1, simian virus 40, f1, and fd DNAs is 5′-T-G-A … 8N … T-G-C-T … 9N … T. However, the sequence in the region of the G4 EcoB site contains an A instead of the final T conserved in these other examples. When the G4 EcoB site is aligned with the other EcoB sites, there are no conserved residues within 50 bases of the common sequence, 5′-T-G-A … 8N … T-G-C-T, except for those seven residues. The analysis of the EcoB site on G4 provides further evidence that only those seven bases are recognized by the E. coli B restriction enzyme.     

The nucleotide sequence recognized by the Escherichia coli K12 restriction and modification enzymes.

The sites recognized by the Escherichia coli K12 restriction endonuclease were localized to defined regions on the genomes of phage φXsK1, φXsK2, and G4 by the marker rescue technique. Methyl groups placed on the genome of plasmid pBR322 by the E. coli K12 modification methylase were mapped in HinfI fragments 1 and 3, and HaeIII fragments 1 and 3. A homology of seven nucleotides in the configuration: 5′-A-A-C .. 6N .. G-T-G-C-3′, where 6N represents six unspecified nucleotides, was found among the DNA sequences containing the five EcoK sites of φXsK1, φXsK2, G4, and pBR322. Three lines of evidence indicate that this sequence constitutes the recognition site of the E. coli K12 restriction enzyme. The C in 5′-A-A-C and the T in 5′-G-T-G-C are locations of mutations leading to loss or gain of the site and thus are positions recognized by the enzyme. This sequence does not occur on φXam3cs70, simian virus 40 (SV40), and fd DNAs which do not possess EcoK sites, and occurs only once on φXsK1, φXsK2, and G4 DNAs, and twice on pBR322 DNA. In order to prove that all seven conserved nucleotides are essential for the recognition by the E. coli K12 restriction enzyme, the nucleotide sequences of φX174, G4, SV40, fd, and pBR322 were searched for sequences differing from the sequence 5′-A-A-C .. 6N .. G-TG-C-3′ at only one of the specified positions. It was found that sequences differing at each of the specified positions occur on DNA sequences that do not contain the EcoK sites. Thus, the recognition site of the E. coli K12 restriction enzyme has the same basic structure as that of the EcoB site (Lautenberger et al., 1978). In each case there are two domains, one containing three and the other four specific nucleotides, separated by a sequence of unspecified bases. However, the unspecified sequence in the EcoK site must be precisely six bases instead of the eight found in the EcoB site. Alignment of the EcoK and EcoB sites suggests that four of the seven specified nucleotides are conserved between the sequences recognized by these two allelic restriction and modification systems.     

A new specific endonuclease from Xanthomonas badrii

A restriction-like endonuelease, XbaI, has been partially purified from Xanthomonas badrii. This enzyme cleaves adenovirus-2 DNA at four sites, bacteriophage lambda DNA at only one site, and does not cleave simian virus 40 DNA. It recognizes the sequence

and cuts at the sites indicated by the arrows.     

Characterization of a rearrangement in viral DNA: mapping of the circular simian virus 40-like DNA containing a triplication of a specific one-third of the viral genome

Serial passage of the non-defective form of a simian virus 40-like virus (DAR) isolated from human brain results in the appearance of three distinct classes of supercoiled DNAs: R_I resistant, R_I sensitive (one cleavage site) and R_I “supersensitive” (three cleavage sites). The R_I cleavage product of the “super sensitive” form is one-third the physical size of simian virus 40 DNA (10.4 S) and reassociates about three times more rapidly than “standard” viral DNA. To identify the portions of the DAR genome present in the 10.4 S segment, the plus strand of each of the 11 fragments of ³²P-labeled simian virus 40 DNA, produced by cleavage with the Hemophilus influenzae restriction endonuclease, was hybridized in solution with the sheared R_I cleavage product of the “supersensitive” class of viral DNA. Reaction was observed with fragments located in two distinct regions of the simian virus 40 genome: (1) Hin-A and C; (2) Hin-G, J, F and K.Further studies indicated that simian virus 40 complementary RNA transcribed in vitro with Escherichia coli RNA polymerase from one strand of simian virus 40 DNA reacts with both strands of the denatured 10.4 S cleavage product when hybridization is monitored with hydroxyapatite. Treatment of the 10.4 S DNA-simian virus 40 cRNA hybrid with the single-strand spcific nuclease, S₁, converted approximately 50% of the radioactive counts to an acid-soluble product. These results indicate that the 10.4 S product contains a transposition of sequences originally present on one of the DAR DNA strands to the other strand. Examination of heteroduplexes formed between the 10.4 S segment and unique linear forms of DAR DNA produced with the R · Eco RI restriction endonuclease have confirmed these observations. Thus it appears that a molecular rearrangement(s) has resulted in the recombination and inversion of viral DNA sequences from two separate loci on the parental DAR genome. This 1.1 × 10⁶ dalton segment is reiterated three times in a supercoiled molecule equivalent in physical size to parental DAR DNA.     

A new specific endonuclease present in Xanthomonas holcicola, Xanthomonas papavericola and Brevibacterium luteum

A new specific endonuclease, XhoI, has been partially purified from Xanthomonas holcicola. This enzyme cleaves adenovirus-2 DNA at five sites, bacteriophage λ DNA at one site, φX174 DNA at one site, but does not cleave simian virus 40 DNA. It recognizes the sequence

and cuts at the sites indicated by the arrows. Enzymes with identical specificity have also been found in Xanthomonas papavericola and Brevibacterium luteum.     

Functional cooperation between exonucleases and endonucleases--basis for the evolution of restriction enzymes

Many types of restriction enzymes cleave DNA away from their recognition site. Using the type III restriction enzyme, EcoP15I, which cleaves DNA 25–27 bp away from its recognition site, we provide evidence to show that an intact recognition site on the cleaved DNA sequesters the restriction enzyme and decreases the effective concentration of the enzyme. EcoP15I restriction enzyme is shown here to perform only a single round of DNA cleavage. Significantly, we show that an exonuclease activity is essential for EcoP15I restriction enzyme to perform multiple rounds of DNA cleavage. This observation may hold true for all restriction enzymes cleaving DNA sufficiently far away from their recognition site. Our results highlight the importance of functional cooperation in the modulation of enzyme activity. Based on results presented here and other data on well-characterised restriction enzymes, a functional evolutionary hierarchy of restriction enzymes is discussed.     

HineI is an isoschizomer of HinfIII restriction endonuclease

HineI is a restriction enzyme isolated from Haemophilus influenzae strain Re. Like other type III restriction endonucleases it requires ATP for cleavage and S-adenosyl-methionine for methylation of DNA. This enzyme recognises the same sequence as HinfIII (Piekarowicz et al., 1981) and cleaves and methylates DNA in a manner similar to all type III restriction enzymes.     

The EcoRI site of simian virus 40 deoxyribonucleic acid: nucleotide sequences of the minus and plus strands.

The nucleotide sequences of the minus and plus strands of simian virus 40 DNA in a 17-base-pair segment spanning the EcoRI restriction site have been identified. The minus-strand sequence is (5′) T-G-G-C-G-A-G-A-A-T-T-C-C-T-T-T-G and the plus-strand sequence is its complement: (5′) C-A-A-A-G-G-A-A-T-T-C-T-C-G-C-C-A.     

A specific endonuclease from Haemophilus haemolyticus.

A restriction-like endonuclease, HhaI, has been partially purified from Haemophilus haemolyticus. This enzyme cleaves bacteriophage lambda DNA and adenovirus-2 DNA at many sites, and cleaves simian virus 40 DNA at only two sites. It recognizes the sequence 5′ -G-C-G-↓C-3′ 3′ -C-↑G-C-G-5′, and cuts at the sites indicated by the arrows.     

Nucleotide sequences at the sites of action of the deoxyribonucleic acid modification enzyme of bacteriophage P1

Labelled oligonucleotides have been fractionated from DNAase digests of phage λ DNA that had been methylated with the phage P1 modification enzyme and S-[methyl-¹⁴C]adenosyl-l-methionine. The longest sequences established are the tetranucleotides pG-ǎ?-T-C4 and p?-T-C-T, which, together with the other sequences determined, particularly pA-G-?, show that the modification enzyme, M.EcoP1, methylates adenine residues within defined sequences and suggest that the oligonucleotide sequence recognized by this enzyme is the hexanucleotide pA-G-A-T-C-T. The duplex formed by base-pairing this hexanucleotide with its complementary sequence resembles the recognition sequence for several restriction enzymes in being bisected by an axis of 2-fold rotational symmetry.     

Role of ATP in the cleavage mechanism of the EcoP15 restriction endonuclease

The EcoP15 restriction endonuclease forms complexes at specific sites on unmodified DNA both in the presence and in the absence of S-adenosyl-l-methionine. ATP acts as an allosteric effector of EcoP15 and induces DNA cleavage followed by release of the enzyme from the DNA. The efficiency of endonucleolytic scission varies from site to site. The nucleotide sequences at sites that are cleaved at a high frequency were compared.     

A specific endonuclease from Brevibacterium albidum

A new restriction-like endonuclease, BalI, has been partially purified from Brevibacterium albidum. This enzyme cleaves bacteriophage λ DNA at least 18 times and adenovirus-2 DNA at least 16 times, but does not cleave simian virus 40 DNA. All sites cleaved by BalI are also cut by the specific endonuclease HaeIII from Haemophilus aegyptius. The recognition sequence of BalI is 5′-T-G-G ↓ C-C-A-3′ 3′-A-C-C ↓ G-G-T-5′ and the cleavage site is indicated by the arrows.     

Restriction and modification enzymes detect no allosteric changes in DNA with bound lac repressor or RNA polymerase

A 203 base-pair fragment containing the lac operator/promoter region of Escherichia coli was inserted into the EcoRI site of the plasmid vector pKC7. Rates of restriction endonuclease cleavage of the flanking EcoRI sites and of several other restriction sites on the DNA molecule were then compared in the presence and absence of bound RNA polymerase or lac repressor. The rates were identical whether or not protein had been bound, even for sites as close as 40 base-pairs from a protein binding site. No difference was detected using supercoiled, nicked circular, or linear DNA substrates. No apparent change in the rates of methylation of EcoRI sites by EcoRI methylase was produced by binding the regulatory proteins.     

Structure–function correlation for the EcoRV restriction enzyme: from non-specific binding to specific DNA cleavage

The EcoRV restriction endonuclease cleaves DNA at its recognition sequence at least a million times faster than at any other DNA sequence. The only cofactor it requires for activity is Mg²⁺: but in binding to DNA in the absence of Mg²⁺, the EcoRV enzyme shows no specificity for its recognition site. Instead, the reason why EcoRV cuts one DNA sequence faster than any other is that the rate of cleavage is controlled by the binding of Mg²⁺ to EcoRV-DNA complexes: the complex at the recognition site has a high affinity for Mg²⁺, while the complexes at other DNA sequences have low affinities for Mg²⁺. The structures of the EcoRV endonuclease, and of its complexes with either 8pecific or non-specific DNA, have been solved by X-ray crystallography. In the specific complex, the protein interacts with the bases in the recognition sequence and the DNA takes up a highly distorted structure. In the non-specific complex with an unrelated DNA sequence, there are virtually no interactions with the bases and the DNA retains a B-like structure. Since the free energy changes for the formation of specific and non-specific complexes are the same, the energy from the specific interactions balances that required for the distortion of the DNA. The distortion inserts the phosphate at the scissile bond into the active site of the enzyme, where it forms part of the binding site for Mg²⁺. Without this distortion, the EcoRV–DNA complex would be unable to bind Mg²⁺ and thus unable to cleave DNA. The specificity of the EcoRV restriction enzyme is therefore governed, not by DNA binding as such, but by its ability to organize the structure of the DNA to which it is bound.     

A new restriction endonuclease Eco31I recognizing a non-palindromic sequence

A restriction endonuclease with a novel site-specificity has been isolated from the Escherichia coli strain RFL31. The nucleotide sequences around a single Eco31I cut on pBR322 DNA and two cuts of λ DNA have been compared. A common ^5′GAGACC_3′CTCTGG sequence occurs near each cleavage site. Precise mapping of the cleavages in both DNA strands places the cuts five nucleotides to the left of the upper sequence and one nucleotide to the left of the lower sequence. This enabled us to deduce the following recognition and cleavage specificity of Eco31I: 5 ′ G G T C T C N ↓ 3 ′ C C A G A G N N N N N ↑     

A third restriction endonuclease from Xanthomonas malvacearum.

An additional sequence-specific endonuclease, XmaIII, has been partially purified from Xanthomonas malvacearum. XmaIII recognizes ten cleavage sites in adenovirus 2 DNA, two sites in bacteriophage lambda and no site in either simian virus 40 DNA or φX174 DNA. It recognizes the sequence

and cleaves at the sites indicated by the arrows. No other endonuclease with this particular nucleotide sequence specificity has been reported.