Characterization of an EcoR124I restriction-modification enzyme produced from a deleted form of the DNA-binding subunit, which results in a novel DNA specificity.

We purified and characterized both the methyltransferase and the endonuclease containing the HsdS delta 50 subunit (type I restriction endonucleases are composed of three subunits--HsdR required for restriction, HsdM required for methylation and HsdS responsible for DNA recognition) produced from the deletion mutation hsdS delta 50 of the type IC R-M system EcoR 124I; this mutant subunit lacks the C-terminal 163 residues of HsdS and produces a novel DNA specificity. Analysis of the purified HsDs delta 50 subunit indicated that during purification it is subject to partial proteolysis resulting in removal of approximately 1 kDa of the polypeptide at the C-terminus. This proteolysis prevented the purification of further deletion mutants, which were determined as having a novel DNA specificity in vivo. After biochemical characterization of the mutant DNA methyltransferase (MTase) and restriction endonuclease we found only one difference comparing with the wild-type enzyme--a significantly higher binding affinity of the MTase for the two substrates of hemimethylated and fully methylated DNA. This indicates that MTase delta 50 is less able to discriminate the methylation status of the DNA during its binding. However, the mutant MTase still preferred hemimethylated DNA as the substrate for methylation. We fused the hsdM and hsdS delta 50 genes and showed that the HsdM-HsdS delta 50 fusion protein is capable of dimerization confirming the model for assembly of this deletion mutant.

A new yeast metabolon involving at least the two first enzymes of arginine biosynthesis: acetylglutamate synthase activity requires complex formation with acetylglutamate kinase.

Open reading frame YJL071W of Saccharomyces cerevisiae was shown to be ARG2 and identified as the structural gene for acetylglutamate synthase, first step in arginine biosynthesis. The three Ascomycete acetylglutamate synthases characterized to date appear homologous, but unlike the other enzymes of the yeast arginine biosynthesis pathway, they showed no significant similarity to their prokaryotic equivalents. The measured synthase activity did not increase with the number of ARG2 gene copies unless the number of ARG5,6 gene copies was increased similarly. ARG5,6 encodes a precursor that is maturated in the mitochondria into acetylglutamate kinase and acetylglutamyl-phosphate reductase, catalyzing the second and third steps in the pathway. The results imply that the synthase must interact stoichiometrically in vivo with the kinase, the reductase, or both to be active. Results obtained with synthetic ARG5 and ARG6 genes suggested that both the kinase and the reductase could be needed. This situation, which has completely escaped notice in yeast until now, is reminiscent of the observation in Neurospora crassa that nonsense arg-6 kinase/reductase mutants lack synthase activity (Hinde, R. W., Jacobson, J. A., Weiss, R. L., and Davis, R. H. (1986) J. Biol. Chem. 261, 5848-5852). In immunoprecipitation experiments, hemagglutinin-tagged synthase coprecipitated with a protein proven by microsequencing to be the kinase. Western blot analyses showed that the synthase has reduced stability in the absence of the kinase/reductase. Our data demonstrate the existence of a new yeast arginine metabolon involving at least the first two, and possibly the first three, enzymes of the pathway. Hypotheses regarding the biological significance of this interaction are discussed.

The yeast ARG7 gene product is autoproteolyzed to two subunit peptides, yielding active ornithine acetyltransferase.

Yeast ornithine acetyltransferase has been purified from total yeast extracts as a heterodimer of two subpeptides (Liu, Y., Van Heeswijck, R., Hoj, P., and Hoogenraad, N. (1995) Eur. J. Biochem. 228, 291-296), confirmed to derive from a single ARG7-encoded precursor (Crabeel, M., Abadjieva, A., Hilven, P., Desimpelaere, J., and Soetens, O. (1997) Eur. J. Biochem. 250, 232-241). By Western immunoblotting, we show that Arg7p is also present as two subpeptides in isolated mitochondria, but that processing occurs before targeting to the mitochondria: deletion of the N-terminal leader peptide results in cytosolic accumulation of N-Arg7p, whereas C-Arg7p partially reaches the organelle by itself. When artificially co-expressed from separate genes, the two subpeptides can complement an arg7 mutation; ornithine acetyltransferase activity is measurable. Maturation of Arg7p occurs at threonine 215 (N-side), in the region most conserved among the 17 ornithine acetyltransferases characterized. Changing this conserved residue to alanine completely abolishes maturation. Furthermore, Arg7p is both processed and active in Escherichia coli, a heterologous background, and is also cleaved in vitro when produced by coupled transcription/translation in a reticulocyte lysate. Together, these data suggest classic autoproteolysis initiated by threonine 215. Most importantly, maturation is required for the enzyme to be functional, since the T215A substitution mutant is catalytically inactive and incapable of genetic complementation, despite its correct targeting to the mitochondria.

A strong carbon source effect is mediated by pUC plasmid sequences in a series of classical yeast vectors designed for promoter characterization.

While using YIp356 and YEp356R lacZ reporter plasmids, we found lacZ expression driven by the ARG2 promoter to be much higher in cells grown on a non-glucose carbon source than in glucose-grown cells (5-10-fold higher on galactose and up to 40-fold higher on ethanol). Furthermore, expression increased 30-fold upon shifting from a high-glucose to a low-glucose medium. This carbon source regulation requires Snf1p and possibly Ssn6p. It appears, however, to be artefactually mediated by plasmid sequences located upstream from the multicloning site. This emerged from the following observations: (a) the derepressive effect disappears if any extra piece of DNA is inserted upstream from the ARG2 promoter; and (b) similar derepression on low glucose is observed with another yeast promoter (ARG11), provided that the flanking 5' region is short. We determined that the cis-elements responsible for this physiologically irrelevant glucose regulation are located between positions 636 and 879 of the pUC18 DNA sequence.

The HsdR subunit of R.EcoR124II: cloning and over-expression of the gene and unexpected properties of the subunit.

Type I restriction endonucleases are composed of three subunits, HsdR, HsdM and HsdS. The HsdR subunit is absolutely required for restriction activity; while an independent methylase is composed of HsdM and HsdS subunits. DNA cleavage is associated with a powerful ATPase activity during which DNA is translocated by the enzyme prior to cleavage. The presence of a Walker type I ATP-binding site within the HsdR subunit suggested that the subunit may be capable of independent enzymatic activity. Therefore, we have, for the first time, cloned and over-expressed the hsdRgene of the type IC restriction endonuclease EcoR124II. The purified HsdR subunit was found to be a soluble monomeric protein capable of DNA- and Mg2+-dependent ATP hydrolysis. The subunit was found to have a weak nuclease activity both in vivo and in vitro, and to bind plasmid DNA; although was not capable of binding a DNA oligoduplex. We were also able to reconstitute the fully active endonuclease from purified M. EcoR124I and HsdR. This is the first clear demonstration that the HsdR subunit of a type I restriction endonuclease is capable of independent enzyme activity, and suggests a mechanism for the evolution of the endonuclease from the independent methylase.

Characterization of the Saccharomyces cerevisiae ARG7 gene encoding ornithine acetyltransferase, an enzyme also endowed with acetylglutamate synthase activity.

We have cloned by functional complementation and characterized the yeast ARG7 gene encoding mitochondrial ornithine acetyltransferase, the enzyme catalyzing the fifth step in arginine biosynthesis. While forming ornithine, this enzyme regenerates acetylglutamate, also produced in the first step by the ARG2-encoded acetylglutamate synthase. Interestingly, total deletion of the genomic ARG7 ORF resulted in an arginine-leaky phenotype, indicating that yeast cells possess an alternative route for generating ornithine from acetylornithine. Yeast ornithine acetyltransferase has been purified and characterized previously as a heterodimer of two subunits proposed to derive from a single precursor protein [Liu, Y-S., Van Heeswijck R., Hoj, P. & Hoogenraad, N. (1995) Eur. J. Biochem. 228, 291-296]; those authors further suggested that the internal processing of Arg7p, which is a mitochondrial enzyme, might occur in the matrix, while the leader peptide would be of the non-cleavable-type. The characterization of the gene (a) establishes that Arg7p is indeed encoded by a single gene, (b) demonstrates the existence of a cleaved mitochondrial prepeptide of eight residues, and (c) shows that the predicted internal processing site is unlike the mitochondrial proteolytic peptidase target sequence. Yeast Arg7p shares between 32-43% identity in pairwise comparisons with the ten analogous bacterial ArgJ enzymes characterized. Among these evolutionarily related enzymes, some but not all appear bifunctional, being able to produce acetylglutamate not only from acetylornithine but also from acetyl-CoA, thus catalyzing the same reaction as the apparently unrelated acetylglutamate synthase. We have addressed the question of the bifunctionality of the eucaryotic enzyme, showing that overexpressed ARG7 can complement yeast arg2 and Escherichia coli argA mutations (affecting acetylglutamate synthase). Furthermore, Arg7p-linked acetylglutamate synthase activity was measurable in an assay. The yeast enzyme is thus clearly, albeit modestly, bifunctional. As with several bacterial ornithine acetyltransferases, the activity of Arg7p was practically insensitive to arginine but strongly inhibited by ornithine, which behaved as a competitive inhibitor.

The type I restriction endonuclease R.EcoR124I: over-production and biochemical properties.

In this paper we describe a two-plasmid system which allows over-production of the R.EcoR124I restriction endonuclease. The endonuclease has been purified to homogeneity in milligram amounts and has been shown to be fully active for both restriction and modification. Unexpectedly, the enzyme was found to require only ATP and Mg2+ for ATPase activity and DNA cleavage; S-adenosyl methionine (SAM), which has been described as a cofactor of type I restriction enzymes, is not required by R.EcoR124I. However, SAM was found to stimulate the rate of ATPase activity and DNA cleavage. This may occur through an increase in specific DNA binding by R.EcoR124I in the presence of SAM, as indicated by our surface plasmon resonance experiments. These functional differences from the well described R.EcoKI restriction endonuclease are reflected in a possible structural difference between the two enzymes, namely that the stoichiometry of R.EcoR124I appears to be R1M2S1 while that of R.EcoKI is R2M2S1. Supercoiled DNA with one or two SR124I recognition sites is cleaved by the same mechanism inferring co-operation between specifically bound and excess enzymes. Nicked-circle DNA is an intermediate of cleavage reaction. Cleavage of DNA was inhibited by an increased degree of negative supercoiling, which may reflect an increased difficulty for the enzyme to translocate the DNA. Hemi-methylated DNA was the preferred substrate for methylation.

Deletions within the DNA recognition subunit of M.EcoR124I that identify a region involved in protein-protein interactions between HsdS and HsdM.

The DNA recognition subunit (HsdS) of type I restriction endonucleases can be divided into domains by means of amino acid identity between subunits from the same family. It has been proposed that DNA-protein interactions occur within the variable domains of the subunit and that protein-protein interactions involve the conserved domains. We have constructed a number of deletion mutants of HsdS that have allowed us to investigate protein-protein interactions. Using a combination of a "competitive" complementation assay and the ability of HsdM to "solubilize" HsdS, we have defined a region within the central conserved domain of HsdS that is responsible for HsdS-HsdM interaction. Computer analysis of amino acid identity between the N-terminal half and the C-terminal half of HsdS identifies a region (repeated in both conserved domains), one copy of which overlaps the region we have identified as essential for HsdS-HsdM interactions, which may be responsible for such protein-protein interactions.

A deletion mutant of the type IC restriction endonuclease EcoR1241 expressing a novel DNA specificity.

We have developed a complementation assay which allows us to distinguish between mutations affecting subunit assembly and mutations affecting DNA binding in the DNA recognition subunit (HsdS) of the multimeric restriction endonuclease EcoR1241. A number of random point mutations were constructed to test the validity of this assay. Two of the mutants produced were found to be truncated polypeptides that were still capable of complementation with the EcoR1241 Hsd subunits to give an active restriction enzyme of novel DNA specificity. The N-terminal variable domain (responsible for recognition of GAA from the EcoR1241 recognition sequence GAAnnnnnnRTCG) and the spacer region (central conserved region) is intact in both of these mutants. One of these mutant genes (hsdS(delta 50) has been cloned as an active Mtase. Purification of the Mtase proved to be difficult because the complex is weak. However, Mtase activity was obtained from a soluble cell extract, and this allowed us to determine the DNA recognition sequence of the Mtase to be GAAnnnnnnnTTC. This recognition sequence is an inverted repeat of 5'-end of the EcoR1241 recognition sequence. This suggests that the mutant Mtase is assembled from two inverted HsdS(D50) subunits, possibly held together by the HsdM subunits.

M13 DNA fingerprinting, a new tool for classification and identification of Lactobacillus spp.

The optimal conditions for the application of M13 DNA fingerprinting to the genus Lactobacillus were determined. Comparative fingerprint analysis of representative strains of Lactobacillus delbrueckii subsp. delbrueckii, Lact. delbrueckii subsp. lactis, Lact. delbrueckii subsp. bulgaricus, Lact. helveticus and Lact. casei permitted the differentiation of species, subspecies and individual strains and the quantitative determination of their genetic relatedness. The results confirm the high specificity of M13 DNA fingerprinting and indicate that it might be used in the classification of Lactobacillus spp.

Genomic variations in mosquitocidal strains of Bacillus sphaericus detected by M13 DNA fingerprinting.

The genomic variation of Bacillus sphaericus reference and local strains belonging to different serotypes was examined by DNA fingerprinting. A phage M13 DNA probe detected a number of variable fragments in the restriction digests of total strain DNAs. The patterns of band distribution showed a certain homology among mosquitocidal strains, expressed by similarity index D and might be a reliable criterion for assessing the level of genomic similarity between closely related strains. An important advantage of DNA fingerprinting is the differentiation of one bacterial strain from another, both expressing common phenotype and possessing highly similar genomic portions. The strain variation revealed by the M13 probe will be useful for characterization of individual strains within a serotype. It could help as well to solve some uncertain cases based on the results obtained by other methods of identification.

DNA fingerprinting of the mosquito pathogen Bacillus sphaericus with M13 DNA as a probe.

Hypervariable nucleotide sequences were detected in Bacillus sphaericus by hybridization with radioactively labelled M13 DNA. Different serotypes could be distinguished by their hybridization profiles. The appearance of bands common for mosquito-pathogenic strains and their absence in an apathogenic strain opens the probability that M13 could hybridize to specific alleles, related to insect toxicity.