In vitro synthesis of double-stranded DNA from the Kilham rat virus single-stranded DNA genome.

Double-stranded, full-length linear DNA was synthesized in vitro by using single-stranded linear DNA as a self-priming template from the parvovirus Kilham rat virus and Escherichia coli DNA polymerase "large fragment" as the polymerizing enzyme. To ascertain the order of the synthesis of the cleavage fragments and to assess the accuracy of the in vitro synthesis, restriction endonuclease cleavage sites with known recognition sequences were mapped on the DNA. Comparing the cleavage pattern of the synthesized DNA with that of double-stranded viral DNA isolated from infected cells confirms that the in vitro synthesis produces a faithful copy of the viral single-stranded genome. Electron micrographs of the in vitro product reveal it to be a double-stranded linear molecule.     

BspLS2I--a new site-specific endonuclease from the thermophilic bacteria Bacillus species LS2]

A new restriction endonuclease BspLS2I was isolated from the thermophilic bacterium Bacillus species LS2 and purified by blue sepharose and hydroxyapatite chromatographies. The enzyme is an isoschizomer of SduI from Streptococcus durans. BspLS2I recognizes the sequence 5' G(G/A/T)GC(C/T/A) decreases C 3' on double-stranded DNA and cleaves it is indicated by the arrow to yield sticky-ended DNA fragments. Maximum catalytic activity of endonuclease was found in 10 mM tris-HCl (pH 7.9) in the presence of 15-30 mM MgCl2 at 50 degrees C. The phage T4 glucosylated DNA is not cleaved by the enzyme.     

Restriction enzyme BsoBI-DNA complex: a tunnel for recognition of degenerate DNA sequences and potential histidine catalysis

BACKGROUND: Restriction endonucleases form a diverse family of proteins with substantial variation in sequence, structure, and interaction with recognition site DNA. BsoBI is a thermophilic restriction endonuclease that exhibits both base-specific and degenerate recognition within the sequence CPyCGPuG. RESULTS: The structure of BsoBI complexed to cognate DNA has been determined to 1.7 A resolution, revealing several unprecedented features. Each BsoBI monomer is formed by inserting a helical domain into an expanded EcoRI-type catalytic domain. DNA is completely encircled by a BsoBI dimer. Recognition sequence DNA lies within a 20 A long tunnel of protein that excludes bulk solvent. Interactions with the specific bases are made in both grooves through direct and water-mediated hydrogen bonding. Interaction with the degenerate position is mediated by a purine-specific hydrogen bond to N7, ensuring specificity, and water-mediated H bonding to the purine N6/O6 and pyrimidine N4/O4, allowing degeneracy. In addition to the conserved active site residues of the DX(n)(E/D)ZK restriction enzyme motif, His253 is positioned to act as a general base. CONCLUSIONS: A catalytic mechanism employing His253 and two metal ions is proposed. If confirmed, this would be the first example of histidine-mediated catalysis in a restriction endonuclease. The structure also provides two novel examples of the role of water in protein-DNA interaction. Degenerate recognition may be mediated by employing water as a hydrogen bond donor or acceptor. The structure of DNA in the tunnel may also be influenced by the absence of bulk solvent.     

Elongation of palindromic repetitive DNA by DNA polymerase from hyperthermophilic archaea: a mechanism of DNA elongation and diversification

DNA polymerase from hyperthermophilic bacteria can elongate tandem repetitive oligoDNA with a complete or incomplete palindromic sequence under isothermal conditions by "hairpin elongation". However, the product of the reaction has not yet been sufficiently characterized. Here, I demonstrate that when palindromic repetitive oligoDNA, e.g., (5'AGATATCT3')(6), was added as a "seed" to the DNA synthesis reaction catalyzed by DNA polymerase from the archaea Thermococcus litoralis (Vent polymerase) at 74 degrees C, the product was (5'AGATATCT3')(n). The product itself was palindromic and repetitive, and its motif (unit) sequence was exactly the same as that of the seed oligoDNA. On the other hand, when a pseudopalindrome, which contains a palindrome-breaking nucleotide (underlined), was present in seed oligoDNA, e.g., (5'GATTC3')(6), the product was (5'GATATC3')(n), which had a different motif sequence from that of the seed oligoDNA. When a pseudopalindrome (5'AGATATCA3')(6) was added to the reaction, the products were 5'TATCA . (AGATATCA)(3) . AGATATCT . (TGATATCT)(5) . TGATA3', etc. When 5'AGATATCA . (AGATATCT3')(5) was added, products were 5'TATCT . (AGATATCT)(2).TGATATCT . AGATATCT . AGATATCA . AGATATCT . AGA3', etc., demonstrating the generation of many "mutations" in the product DNA. I conclude that a tandem repetitive sequence is faithfully elongated (amplified) by hyperthermophilic DNA polymerase if it is completely palindromic, but is elongated with many errors if it is incompletely palindromic (pseudopalindromic) or mixed with a pseudopalindrome. The results suggest a protein-catalyzed elongation/diversification mechanism of short repetitive DNAs on the early earth.     

Determination of DNA sequences containing methylcytosine in Bacillus subtilis Marburg.

The methylcytosine-containing sequences in the DNA of Bacillus subtilis 168 Marburg (restriction-modification type BsuM) were determined by three different methods: (i) examination of in vivo-methylated DNA by restriction enzyme digestion and, whenever possible, analysis for methylcytosine at the 5' end; (ii) methylation in vitro of unmethylated DNA with B. subtilis DNA methyltransferase and determination of the methylated sites; and (iii) the methylatability of unmethylated DNA by B. subtilis methyltransferase after potential sites have been destroyed by digestion with restriction endonucleases. The results obtained by these methods, taken together, show that methylcytosine was present only within the sequence 5'-TCGA-3'. The presence of methylcytosine at the 5' end of the DNA fragments generated by restriction endonuclease AsuII digestion and the fact that in vivo-methylated DNA could not be digested by the enzyme XhoI showed that the recognition sequences of these two enzymes contained methylcytosine. As these two enzymes recognized a similar sequence containing a 5' pyrimidine (Py) and a 3' purine (Pu), 5'-PyTCGAPu-3', the possibility that methylcytosine is present in the complementary sequences 5'-TTCGAG-3' and 5'-CTCGAA-3' was postulated. This was verified by the methylation in vitro, with B. subtilis enzyme, of a 2.6-kilobase fragment of lambda DNA containing two such sites and devoid of AsuII or XhoI recognition sequences. By analyzing the methylatable sites, it was found that in one of the two PyTCGAPu sequences, cytosine was methylated in vitro in both DNA strands. It is concluded that the sequence 5'-PyTCGAPu-3' is methylated by the DNA methyltransferase (of cytosine) of B. subtilis Marburg.     

A general method to cleave a known DNA sequence at any site.

We describe a new method for obtaining DNA fragments starting at a desired point where there is no recognition sequence for any known restriction endonuclease. A single-stranded DNA containing the fragment of interest is annealed to a synthetic oligonucleotide hybridizing at the 5' end of the required fragment. Then, a partially double-stranded DNA is synthesized using the Klenow fragment of DNA polymerase I in the presence of the four deoxynucleoside triphosphates. The remaining single-stranded regions are removed by digestion with a single-strand nuclease, and the resulting 5' blunt-ended fragment is finally released by digestion with a restriction endonuclease at any site downstream its 3' end. The usefulness of the method was exemplified here by insertion of an epidermal growth factor-like African swine fever virus gene immediately downstream of the ribosome binding site of an expression vector.     

Nontemplate polymerization of free nucleotides into genetic elements by thermophilic DNA polymerase in vitro

DNA synthesis is the cornerstone of all life forms and is required to replicate and restore the genetic information. Usually, DNA synthesis is carried out only by DNA polymerases semiconservatively to copy preexisting DNA templates. We report here that DNA strands were synthesized ab initio in the absence of any DNA or RNA template by thermophilic DNA polymerases at (a) a constant high temperature (74°C), (b) alternating temperatures (94°C/60°C/74°C), or (c) physiological temperatures (37°C). The majority of the ab initio synthesized DNA represented short sequence blocks, repeated sequences, intergenic spacers, and other unknown genetic elements. These results suggest that novel DNA elements could be synthesized in the absence of a nucleic acid template by thermophilic DNA polymerases in vitro. Biogenesis of genetic information by thermophilic DNA polymerase-mediated nontemplate DNA synthesis may explain the origin of genetic information and could serve as a new way of biosynthesis of genetic information that may have facilitated the evolution of life.     

Site-specific mutagenesis method which completely excludes wild-type DNA from the transformants.

A highly efficient site-specific mutagenesis method has been devised to exclude wild-type DNA from incorporation into the transformed cells. Two complementary oligonucleotides, corresponding to a target sequence of a DNA molecule and containing an insertion mutation which created an endonuclease restriction site, were synthesized. By using the wild-type DNA molecule flanked by two restriction sites on each side of the target region as a template, the two oligonucleotide primers were extended, enriched, and isolated. The extended products, in turn, were used as templates in a polymerase chain reaction to obtain a mutagenized double-stranded DNA fragment which was conveniently cloned into plasmids by using the flanking restriction sites. Escherichia coli cells transformed by these plasmids were subject to large-scale analysis. One hundred percent of the transformants examined by colony hybridization, restriction enzyme analysis, and DNA sequencing were found to contain the mutant DNA sequence.     

Site-specific mutagenesis method which completely excludes wild-type DNA from the transformants.

A highly efficient site-specific mutagenesis method has been devised to exclude wild-type DNA from incorporation into the transformed cells. Two complementary oligonucleotides, corresponding to a target sequence of a DNA molecule and containing an insertion mutation which created an endonuclease restriction site, were synthesized. By using the wild-type DNA molecule flanked by two restriction sites on each side of the target region as a template, the two oligonucleotide primers were extended, enriched, and isolated. The extended products, in turn, were used as templates in a polymerase chain reaction to obtain a mutagenized double-stranded DNA fragment which was conveniently cloned into plasmids by using the flanking restriction sites. Escherichia coli cells transformed by these plasmids were subject to large-scale analysis. One hundred percent of the transformants examined by colony hybridization, restriction enzyme analysis, and DNA sequencing were found to contain the mutant DNA sequence.     

TspGWI, a thermophilic class-IIS restriction endonuclease from Thermus sp., recognizes novel asymmetric sequence 5'-ACGGA(N11/9)-3'

A novel prototype class-IIS restriction endonuclease, TspGWI, was isolated from the thermophilic bacterium Thermus sp. GW. The recognition sequence and cleavage positions have been established: TspGWI recognizes the non-palindromic 5-bp sequence 5'-ACGGA-3' and cleaves the DNA 11 and 9 nt downstream in the top and bottom strand, respectively. In addition, an accompanying endonuclease, TspGWII, an isoschizomer of Pst I, was found in Thermus sp. GW cells.     

Isolation and characterisation of DraI, a type II restriction endonuclease recognising a sequence containing only A:T basepairs, and inhibition of its activity by uv irradiation of substrate DNA

A type II restriction endonuclease, DraI, isolated from Deinococcus radiophilus ATCC 27603 recognises the palindromic hexanucleotide sequence (formula; see text) and cleaves it, as indicated by the arrows, to produce blunt-ended fragments. The yield of enzyme is 100 to 1000 times that of the only other known type II restriction endonuclease that recognises a sequence composed solely of A:T basepairs, the isoschizomer AhaIII (1). Ultraviolet irradiation of the DNA substrate at relatively low doses inhibits the activity of DraI by "protecting" the recognition sequence and this may be exploited to give control of partial digestion of DNA by DraI.     

Structure of Hepatitis B Dane Particle DNA and Nature of the Endogenous DNA Polymerase Reaction

The circular DNA of hepatitis B Dane particles, which serves as the primer/template for an endogenous DNA polymerase, was analyzed by electrophoresis before and after a polymerase reaction and after digestion by restriction endonuclease or single-strand-specific endonuclease S1. The unreacted molecules extracted from the particles were electrophoretically heterogeneous, and treatment with S1 nuclease produced double-stranded linear DNA ranging in length from 1,700 to 2,800 base pairs (bp). After an endogenous DNA polymerase reaction, two discrete species of DNA molecules were found: a circular form and a linear form 3,200 bp long. The reaction resulted in a population of molecules with an elongated and more homogeneous double-stranded region. These results suggest that the circular molecules in Dane particles have single-stranded regions of varying lengths that are made double stranded during the DNA polymerase reaction. The endogenous DNA polymerase was found to initiate apparently at random in a region spanning more than a third of the molecule. Analysis of restriction endonuclease cleavage fragments of the fully elongated DNA revealed that although the molecules were of a uniform length, they were somewhat heterogeneous in sequence. The sum of the sizes of the 10 major endonuclease Hae III-generated fragments, detected by ethidium bromide, was 3,880 bp. Two additional fragments (B and G) detected by autoradiography after an endogenous DNA polymerase reaction with (32)P-labeled deoxynucleoside triphosphates made the total 4,910 bp.     

Some combinatorial problems of DNA restriction fragment length polymorphisms

Recombinant DNA techniques provide a means of defining new polymorphisms at the DNA sequence level. Polymorphisms arise when individuals differ in the location and number of sites where restriction endonucleases can cleave their DNA. Each such site exhibits two possible states: one for the presence of a specific endonuclease recognition sequence, the other for its absence. The states of a system of adjacent sites can be revealed experimentally by cleaving a person's DNA into a set of fragments. For experimentally well-understood systems of sites, we consider problems of counting numbers of possible fragments, haplotypes, genotypes, and phenotypes, and the means of resolving phenotype-genotype ambiguities. The degree of polymorphism generated by such systems and the importance to gene mapping are discussed.     

Bidirectional sequencing of supercoiled plasmid DNA

In this paper we show that restriction DNA fragments can prime DNA synthesis of a homologous supercoiled plasmid DNA. Using the dideoxyribonucleotide chain terminator method, newly synthesized truncated chains can be detached from the primers by restriction enzyme digestion. Therefore, by choosing DNA fragments flanked by two different restriction enzymes sites, nucleotide sequence information can be simultaneously obtained on both regions of the DNA surrounding the restriction fragment. The advantage of this sequencing approach over current methods is that no prior knowledge of the primary sequence is needed to find the nucleotide sequence of a given DNA fragment. Thus, synthetic primers are not required and internal sequences of a given clone can be easily accessed without the need of fragmenting the original construct. The method has been used with rapid plasmid preparations, thus considerable time and effort can be saved in the gathering of nucleotide sequence information.     

Multi-priming sequencing: a DNA sequencing method involving restriction enzyme-digested DNA fragments as primers.

An improved strategy for fluorescence-labeled dideoxy chain termination sequencing involving restriction enzyme-digested DNA fragments as primers, which are prepared from the DNA to be sequenced, is described. By using modified nucleoside triphosphates for strand protection in chain termination reactions, newly synthesized chains were detached from a primer at the regenerated recognition site by means of suitable restriction enzyme digestion. The digests could be analyzed with commercial automated DNA sequencers. Thus, by using restriction DNA fragments (double-stranded) as primers, sequence information was obtained from both "minus" and "plus" single-stranded DNA templates without subcloning. Nor is the synthesis of oligonucleotide primers needed. This method, named "Multi-Priming Sequencing," was proven to be time-saving, economical, and effective compared to conventional methods.     

RSITE: a computer program to predict the recognition sequence of a restriction enzyme.

A computer program (RSITE) was developed which predicts the recognition sequence of a restriction endonuclease. The sizes of fragments experimentally determined on cleavage of a DNA of known sequence were input. Possible recognition sequences producing fragments of sizes matching those determined empirically were printed out. The program faithfully predicted the specificity of restriction enzymes of known recognition sequence and also determined the recognition sequence of a new restriction enzyme from Haemophilus influenzae GU (HinGU II).     

Crystallization of complexes of EcoRV endonuclease with cognate and non-cognate DNA fragments

Complexes of the type II restriction endonuclease EcoRV with a variety of short, selfcomplementary deoxyoligonucleotides have been crystallized. The best crystals diffract to about 2.7 A resolution and consist of 1:1 complexes between endonuclease dimers and duplexes of the cognate decamer GGGATATCCC containing the hexameric RV recognition sequence GATATC. Crystals with the non-cognate DNA octamer duplexes CGAGCTCG and CGAATTCG diffract to 3.0 and 3.5 A resolution, respectively, and contain two DNA duplexes per enzyme dimer.     

Recognition sequence of restriction endonuclease KpnI from Klebsiella pneumoniae.

We have determined the recognition sequence of the restriction endonuclease KpnI, previously isolated from Klebsiella pneumoniae. The enzyme cleaves the twofold rotationally symmetric sequence (see book for formula) at the positions indicated by the arrows, producing 3' protruding cohesive ends, four nucleotides in length. The specific cleavage site was unambiguously deduced using both 3' and 5' end analyses of KpnI generated restriction fragments of simian-virus 40 (SV40) DNA (1 site), adenovirus-2 (Ad-2) DNA (8 sites), and a plasmid (pCRI) DNA (2 sites).     

Creating seamless junctions independent of restriction sites in PCR cloning

A method is described for the efficient cloning of any given DNA sequence into any desired location without the limitation of naturally occurring restriction sites. The technique employs the polymerase chain reaction (PCR) combined with the capacity of the type-IIS restriction endonuclease (ENase) Eam1104I to cut outside its recognition sequence. Primers that contain the Eam1104I recognition site (5′-CTCTTC) are used to amplify the DNA fragments being manipulated. Because the ENase is inhibited by site-specific methylation in the recognition sequence, all internal Eam1104I sites present in the DNA can be protected by performing the PCR amplification in the presence of 5-methyl-deoxycytosine (^m5dCTP). The primer-encoded Eam1104I sites are not affected by the modified nucleotides (nt) since the newly synthesized strand does not contain any cytosine residues in the recognition sequence. In addition, the ENase's ability to cleave several bases downstream from its recognition site allows the removal of superfluous, terminal sequences from the amplified DNA fragments, resulting in 5′ overhangs that are defined by the nt present within the cleavage site. Thus, the elimination of extraneous nt and the generation of unique, non-palindromic sticky ends permits the formation of seamless junctions in a directional fashion during the subsequent ligation event.