Activity-based in vitro selection of T4 DNA ligase

Recent in vitro methodologies for selection and directed evolution of proteins have concentrated not only on proteins with affinity such as single-chain antibody but also on enzymes. We developed a display technology for selection of T4 DNA ligase on ribosome because an in vitro selection method for DNA ligase had never been developed. The 3' end of mRNA encoding the gene of active or inactive T4 DNA ligase-spacer peptide fusion protein was hybridized to dsDNA fragments with cohesive ends, the substrate of T4 DNA ligase. After in vitro translation of the mRNA-dsDNA complex in a rabbit reticulocyte system, a mRNA-dsDNA-ribosome-ligase complex was produced. T4 DNA ligase enzyme displayed on a ribosome, through addition of a spacer peptide, is able to react with dsDNA in the complex. The complex expressing active ligase was biotinylated by ligation with another biotinylated dsDNA probe and selected with streptavidin-coated magnetic beads. We effectively selected active T4 DNA ligase from a small amount of protein. The gene of the active T4 DNA ligase was enriched 40 times from a mixture of active and inactive genes using this selection strategy. This ribosomal display strategy may have high potential to be useful for selection of other enzymes associated with DNA.     

The MmeI family: type II restriction–modification enzymes that employ single-strand modification for host protection

The type II restriction endonucleases form one of the largest families of biochemically-characterized proteins. These endonucleases typically share little sequence similarity, except among isoschizomers that recognize the same sequence. MmeI is an unusual type II restriction endonuclease that combines endonuclease and methyltransferase activities in a single polypeptide. MmeI cuts DNA 20 bases from its recognition sequence and modifies just one DNA strand for host protection. Using MmeI as query we have identified numerous putative genes highly similar to MmeI in database sequences. We have cloned and characterized 20 of these MmeI homologs. Each cuts DNA at the same distance as MmeI and each modifies a conserved adenine on only one DNA strand for host protection. However each enzyme recognizes a unique DNA sequence, suggesting these enzymes are undergoing rapid evolution of DNA specificity. The MmeI family thus provides a rich source of novel endonucleases while affording an opportunity to observe the evolution of DNA specificity. Because the MmeI family enzymes employ modification of only one DNA strand for host protection, unlike previously described type II systems, we propose that such single-strand modification systems be classified as a new subgroup, the type IIL enzymes, for Lone strand DNA modification.     

A nomenclature for restriction enzymes,DNA methyltransferases,homing endonucleases and their genes

A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes and gene products. It provides explicit categories for the many different Type II enzymes now identified and provides a system for naming the putative genes found by sequence analysis of microbial genomes.     

Location of the 5-Methylcytosine Group on the Bacteriophage φX174 Genome

Bacteriophage phiX174 DNA was labeled in vivo with [methyl-(3)H]methionine. The methyl-labeled progeny DNA was extracted from purified bacteriophage phiX174 particles and was used as template for in vitro synthesis of the complementary strand in the presence of the nucleoside triphosphates and Escherichia coli polymerase I. The resultant replicative form DNA was then cleaved, in separate experiments, with restriction endonucleases from Haemophilus influenzae and H. aegyptius. The DNA fragments were analyzed by polyacrylamide gel electrophoresis. It is concluded that the single methylcytosine in the viral DNA is located in a specific region of the phiX174 genome, very likely in gene H.     

Derivation of a restriction map of bacteriophage T3 DNA and comparison with the map of bacteriophage T7 DNA.

The DNA of bacteriophage T3 was characterized by cleavage with seven restriction endonucleases. AvaI, XbaI, BglII, and HindIII each cut T3 DNA at 1 site, KpnI cleaved it at 2 sites, MboI cleaved it at 9 sites, and HpaI cleaved it at 17 sites. The sizes of the fragments produced by digestion with these enzymes were determined by using restriction fragments of T7 DNA as molecular weight standards. As a result of this analysis, the size of T3 DNA was estimated to be 38.74 kilobases. The fragments were ordered with respect to each other and to the genetic map to produce a restriction map of T3 DNA. The location and occurrence of the restriction sites in T3 DNA are compared with those in the DNA of the closely related bacteriophage T7.     

Construction and characterization of new cloning vehicles. I. Ampicillin-resistant derivatives of the plasmid pMB9.

In vitro recombination via restriction endonucleases and the in vivo genetic translocation of the Ap resistance (Apr) gene resulted in the construction of a new cloning vehicle, the plasmid pBR313. This vector was derived from a ColE1-like plasmid and, while it does not produce colicon E1, it still retains colicin E1 immunity. The Apr and tetracycline resistance (Tcr) markers carried in pBR313 were derived from the ampicillin transposon (TnA) of pRSF2124 and pSC101 respectively. During the construction of pBR313, the TnA component was altered and the Apr gene in pBR313 can no longer be translocated. This plasmid has a molecular weight of 5.8 Mdalton and has been characterized using thirteen restriction enzymes, six of which (EcoRI, SmaI, HpaI, HindIII, BamHI and SalI) cleave the plasmid at unique restriction sites. This allows the molecular cloning of DNA fragments generated by these six enzymes. The restriction sites for the latter three enzymes, HindIII, BamHI and SalI, are located in the Tcr gene(s). Cloning DNA fragments into these sites alters the expression of the Tcr mechanisms thus providing a selection for cells carrying recombinant plasmid molecules. An enrichment method for AprTcS cells carrying recombinant plasmid molecules is described.     

Characterization of AloI, a restriction-modification system of a new type.

We report the properties of the new AloI restriction and modification enzyme from Acinetobacter lwoffi Ks 4-8 that recognizes the DNA target 5' GGA(N)6GTTC3' (complementary strand 5' GAAC(N)6TCC3'), and the nucleotide sequence of the gene encoding this enzyme. AloI is a bifunctional large polypeptide (deduced M(r) 143 kDa) revealing both DNA endonuclease and methyltransferase activities. Depending on reaction cofactors, AloI cleaves double-stranded DNA on both strands, seven bases on the 5' side, and 12-13 bases on the 3' side of its recognition sequence, and modifies adenine residues in both DNA strands in the target sequence yielding N6-methyladenine. For cleavage activity AloI maintains an absolute requirement for Mg(2+) and does not depend on or is stimulated by either ATP or S-adenosyl-L-methionine. Modification function requires the presence of S-adenosyl-L-methionine and is stimulated by metal ions (Ca(2+)). The C-terminal and central parts of the protein were found to be homologous to certain specificity (HsdS) and modification (HsdM) subunits of type I R-M systems, respectively. The N-terminal part of the protein possesses the putative endonucleolytic motif DXnEXK of restriction endonucleases. The deduced amino acid sequence of AloI shares significant homology with polypeptides encoding HaeIV and CjeI restriction-modification proteins at the N-terminal and central, but not at the C-terminal domains. The organization of AloI implies that its evolution involved fusion of an endonuclease and the two subunits, HsdM and HsdS, of type I restriction enzymes. According to the structure and function properties AloI may be regarded as one more representative of a newly emerging group of HaeIV-like restriction endonucleases. Discovery of these enzymes opens new opportunities for constructing restriction endonucleases with a new specificity.     

Organization of ribosomal protein genes in Escherichia coli: II. Mapping of ribosomal protein genes by in vitro synthesis of ribosomal proteins using DNA fragments of a transducing phage as templates

The structural genes for six ribosomal proteins (r-proteins) located in the str-spc region around 64 minutes on the Escherichia coli chromosome have been mapped physically with respect to each other and the neighboring genes aroE and trkA. The genes code for the 30 S r-proteins S4 (ram), S5 (spc), S8, S11, S13 and S14. Furthermore, regions coding for unidentified 50 S r-proteins have been indicated.The mapping was performed by biochemical methods employing DNA from the specialized transducing phage λspc1, which carries the aroE-trkA-spc region of the E. coli chromosome. The phage DNA was cleaved by restriction endonucleases, and the generated DNA fragments used as templates for synthesis of r-proteins in a DNA-dependent cell-free system. Since the relative order of the DNA fragments created by the restriction endonucleases is known, a genetic map could be constructed.     

REF Select: expert system software for selecting restriction endonucleases for restriction endonuclease fingerprinting

REF Select, expert system software, has been developed to assist in the selection of optimal restriction endonucleases for restriction endonuclease fingerprinting (REF), a method for rapid and sensitive mutation screening of long DNA segments (1-2 kb). The REF method typically involves six separate digestions with up to two restriction endnonucleases used in each digestion. If done manually, performing a comprehensive review of the large number of possible sets of restriction endonucleases that could be used (over 10(19) in the example presented here) and making an optimal choice is not feasible. Furthermore, the typical nonoptimal manual selection takes approximately 8 h by someone experienced with REF. REF Select enables a comprehensive review of the possible sets and a consistent, objective and fast selection of an optimal set by using a two-step strategy: the selection of sets that meet specific constraints, which is followed by a ranking of those sets by an optimality score. Based on our experience with REF, we chose default selection and ranking parameters to help the user get started quickly. These parameters form a knowledge base that can be customized and then saved by the user. In conclusion, REF Select facilitates the general application of REF by serving as an expert system for the selection of optimal restriction endonucleases. We demonstrated REF Select using an example segment from the human p53 gene.     

Immobilization of restrictases

The search for optimal variants of restriction endonucleases immobilization was begun recently. For some enzymes immobilization was successful due to the presence of covalent bonds on CNBr-sepharose (EcoRI, BamHI, HindIII, TaqI, PaeI, SalI, PvuII). For the enzymes EcoRI, BamHI and HindIII it was due to hydrophobic interaction with triethyl-agarose (triethyl-triphenylmethane). The high yield (up to 80%) of enzymatic activity has been obtained for small number of restriction endonucleases. In the experiments of several amino acid residues modification and immobilization of restriction endonucleases the participation of lysine, arginine, glutamic acid and SH- or S-S-groups in the catalysis and (or) binding of these enzymes with DNA has been shown. The restriction endonucleases immobilization experiments and research of enzymes active centre enrich each other and are very interesting for their use in molecular biology and deepening our knowledge of protein-nucleic interactions.     

DNA recombination in vitro initiated by Ca/Mg-dependent endonuclease from cell nuclei of human splenocytes

The system of DNA recombination in vitro was constructed. It comprises two plasmids, the derivatives of pBR322 deleted in the genes for tetracycline resistance, and the recombinogenic extract of the thymus lymphocytes nuclei of mice. The system permits to study the effect of proteins and factors on the efficiency of recombination resulting in reconstruction of the tetracycline resistance gene. Double-strand cuts in one of the deleted plasmids were necessary for recombination. Double-strand cuts by Ca/Mg-dependent endonuclease of the human spleen lymphocytes nuclei were more efficient as compared with the ones of DNAase I, restriction endonucleases PaeI and SalI in the initiation of recombination. The possible role of Ca/Mg-dependent endonuclease in recombination in vivo is discussed.     

Cloning and analysis of the genes encoding the type IIS restriction-modification system HphI from Haemophilus parahaemolyticus.

The genomic region encoding the type IIS restriction-modification (R-M) system HphI (enzymes recognizing the asymmetric sequence 5'-GGTGA-3'/5'-TCACC-3') from Haemophilus parahaemolyticus were cloned into Escherichia coli and sequenced. Sequence analysis of the R-M HphI system revealed three adjacent genes aligned in the same orientation: a cytosine 5 methyltransferase (gene hphIMC), an adenine N6 methyltransferase (hphIMA) and the HphI restriction endonuclease (gene hphIR). Either methyltransferase is capable of protecting plasmid DNA in vivo against the action of the cognate restriction endonuclease. hphIMA methylation renders plasmid DNA resistant to R.Hindill at overlapping sites, suggesting that the adenine methyltransferase modifies the 3'-terminal A residue on the GGTGA strand. Strong homology was found between the N-terminal part of the m6A methyltransferasease and an unidentified reading frame interrupted by an incomplete gaIE gene of Neisseria meningitidis. The HphI R-M genes are flanked by a copy of a 56 bp direct nucleotide repeat on each side. Similar sequences have also been identified in the non-coding regions of H.influenzae Rd DNA. Possible involvement of the repeat sequences in the mobility of the HphI R-M system is discussed.