UV- and CD-spectra of restriction endonuclease EcoRII and DNA-methylase EcoRII

UV- and CD-spectra of homogeneous enzymes have been measured. Extinction coefficients estimated from the UV-spectra are 0.97 for restriction endonuclease EcoRII at 279.5 nm and 1.17 for DNA-methylase EcoRII at 279 nm. As it follows from the CD spectra, both enzymes have a well developed tertiary structure and a highly ordered secondary structure, which consists of 22% alpha-helices, 64% beta-structure and 9% bends for REcoRII and of 44% alpha-helices, 48% beta-structure and 4% bends for MEcoRII. Restriction endonuclease denatures at 50 degrees C, while DNA-methylase denatures at 45 degrees C, with partial reversibility upon cooling.     

Overproduction of the EcoRII endonuclease and methylase by Escherichia coli strains carrying recombinant plasmids constructed in vitro

Recombinant DNA molecules were constructed from the plasmid pIL203 and the EcoRI-fragment of N3 plasmid containing EcoRII endonuclease and methylase genes and also a gene for resistance to sulfanilamide. The pIL203 plasmid, used as a vector, consisted of the Bam HI-EcoRI-fragment of the plasmid pBR322 conferring resistance to ampicillin and the Bam HI-EcoRI-fragment of lambda phage containing promoters, a thermosensitive mutation in the cI gene and a suppressible amber mutation in the cro gene. Ampicillin-sulfanilamide-resistant clones were selected and tested for their restriction and modification phenotype. The recombinant plasmid DNA, isolated from ApRSuR-resistant clones, which restricted and modified phage lambda imm21 with EcoRII specificity, had the EcoRI-fragment with EcoRII genes in a single orientation. The recombinant plasmid pSK323 was transferred into E. coli strains with su-, su1, su2 or su3 phenotypes. The synthesis of products of EcoRII genes by these strains grown at 37 degrees C is increased by 10--50-fold.     

Molecular cloning and expression in Escherichia coli of two modification methylase genes of Bacillus subtilis

Two modification methylase genes of Bacillus subtilis R were cloned in Escherichia coli by using a selection procedure which is based on the expression of these genes. Both genes code for DNA-methyltransferases which render the DNA of the cloning host E. coli HB101 insensitive to the BspRI (5'-GGCC) endonuclease of Bacillus sphaericus R. One of the cloned genes is part of the restriction-modification (RM) system BsuRI of B. subtilis R with specificity for 5'-GGCC. The other one is associated with the lysogenizing phage SP beta B and produces the methylase M.BsuP beta BI with specificity for 5'-GGCC. The fragment carrying the SP beta B-derived gene also directs the synthesis in E. coli of a third methylase activity (M.BsuP beta BII), which protects the host DNA against HpaII and MspI cleavage within the sequence 5'-CCGG. Indirect evidence suggests that the two SP beta B modification activities are encoded by the same gene. No cross-hybridization was detected either between the M.BsuRI and M.BsuP beta B genes or between these and the modification methylase gene of B. sphaericus R, which codes for the enzyme M.BspRI with 5'-GGCC specificity.     

Binding of the EcoRII methylase to azacytosine-containing DNA.

Binding of DNA(cytosine-5)methyltransferases to azacytosine containing DNA is stimulated by the presence of S-adenosyl-methionine or its analogs sinefungin or S-adenosyl-L-homocysteine. Methylation of the DNA is therefore not necessary for binding to occur. There is no relationship between the affinity of the analog for the EcoRII enzyme and its ability to stimulate binding. The DNA-enzyme complex partially dissociates on incubation in 0.1% sodium dodecyl sulfate and 0.5 M ammonium acetate. Some of this DNA could again form a tight complex with enzyme, indicating that DNA-enzyme complex formation is reversible. Binding occurs when the second cytosine in the sequence CCAGG is substituted by azacytosine. This is the cytosine that would normally be methylated by the enzyme. The binding is therefore due to specific interaction of the methylase with azacytosine at the site it would normally methylate.     

Isolation, purification and properties of restriction endonuclease EcoRII

The restriction endonuclease Eco RII was isolated and purified to homogeneity. The isolation procedure involved the use of the E. coli strain B834/pSK323, containing the recombinant plasmide pSK323 which provides for the oversynthesis of Eco RII enzymes. Data from gel filtration and Na-DS electrophoresis suggest that the restriction endonuclease Eco RII is a protein made up of two subunits, each with molecular weight of 44 000.     

Nucleotide sequence of the EcoRII restriction endonuclease gene

The nucleotide sequence of a 1394 basepair (bp) DNA fragment containing the EcoRII restriction endonuclease (R.EcoRII) gene was determined. The endonuclease gene is 1206 bp in length (predicted 402 amino acids (aa) and Mr = 45 178) and is separated by 33 bp from the EcoRII modification methylase (M.EcoRII) gene. The EcoRII restriction-modification system has a tail-to-tail organization of the two genes.     

Purification of restriction endonuclease EcoRII using monoclonal antibodies

Monoclonal antibodies against EcoRII endonuclease were obtained after immunization of two BALB/c mice with a homogeneous enzyme prepared by conventional methods. IgG from ascitic fluid was purified and coupled to CNBr-activated Sepharose 4B to give a specific column used to isolate EcoRII endonuclease. The isolated EcoRII endonuclease produced a single band during SDS gel electrophoresis.     

Localization of SsoII restriction endonuclease and methylase genes on the map of the P4 plasmid

The restrictional mapping of naturally occurring plasmid P4 from Shigella sonnei 47 strain coding for the SsoII restriction endonuclease and methylase genes has been made. Using the genetic engineering approach the locations of the SsoII host cell specificity system enzymes genes have been determined.     

Overproduction of the EcoR V endonuclease and methylase.

Strains overproducing the EcoR V endonuclease and methylase have been obtained by inserting each of the two genes in expression vectors containing the lambda PL promoter. The methylase is overproduced to a level reaching 5-10% of the total cellular proteins, which represents a 50-100 fold increase. A 30 fold overproduction of endonuclease was achieved by randomly positioning the EndRV gene downstream of the lambda PL promoter. The situation in the endonuclease overproducing clone resembles that encountered in maxi-cells. The strains described here allowed a quick purification of both enzymes in sufficient amounts for crystallisation attempts.     

Inhibition of recA-mediated strand exchange by adducts of azacytosine-containing DNA and the EcoRII methylase

Wild type Escherichia coli cells containing elevated levels of DNA (cytosine-5)methyltransferases have increased sensitivity to the toxic effects of 5-azacytidine. The methyltransferases form tight binding complexes with azacytosine in DNA which could interfere with the recA recBCD repair pathway which is largely responsible for cell survival after treatment with the drug. We therefore determined if these complexes interfered with recA-mediated strand exchange in vitro. 32P-Labeled DNA fragments containing a single EcoRII site, with cytosine in the (-) strand replaced by 5-azacytosine, were prepared. We investigated the effect of the EcoRII methyltransferase on recA-mediated strand exchange with homologous M13 DNA by electrophoresis in agarose gels. In the absence of the methylase the rate and extent of strand exchange of azacytosine-containing DNA is the same as control DNA. In the presence of the methyltransferase strand exchange is inhibited, but some incorporation of duplexes into recA-single-stranded DNA (ssDNA) complexes still occurs. The formation of these complexes is dependent on the length of the fragment 3' to the methylase binding site on the strand complementary to the ssDNA. The greater the length the greater the number of complexes that form. S-Adenosyl-L-methionine, which enhances binding of the methyltransferase to azacytosine-containing DNA, causes an increase in the inhibition of strand exchange and an increase in the number of inactive complexes formed. The complexes can be dissociated with guanidinium chloride which denatures the methyltransferase and leads to release of the (+) strand. The (-) strand remains associated with the ssDNA. This result implies that a plectonemic joint is formed between recA-ssDNA complexes and azacytosine-containing DNA-methyltransferase complexes. However, branch migration in these complexes is inhibited. Denaturation of the methyltransferase allows branch migration to proceed to completion, releasing the (+) strand.     

Molecular analysis of restriction endonuclease EcoRII from Escherichia coli reveals precise regulation of its enzymatic activity by autoinhibition

Bacterial restriction endonuclease EcoRII requires two recognition sites to cleave DNA. Proteolysis of EcoRII revealed the existence of two stable domains, EcoRII-N and EcoRII-C. Reduction of the enzyme to its C-terminal domain, EcoRII-C, unleashed the enzyme activity; this truncated form no longer needed two recognition sites and cleaved DNA much more efficiently than EcoRII wild-type. The crystal structure of EcoRII showed that probably the N-terminal domain sterically occludes the catalytic site, thus apparently controlling the cleavage activity. Based on these data, EcoRII was the first restriction endonuclease for which an autoinhibition mechanism as regulatory strategy was proposed. In this study, we probed this assumption and searched for the inhibitory element that mediates autoinhibition. Here we show that repression of EcoRII-C is achieved by addition of the inhibitory domain EcoRII-N or by single soluble peptides thereof in trans . Moreover, we perturbed contacts between the N- and the C-terminal domain of EcoRII by site-directed mutagenesis and proved that β-strand B1 and α-helix H2 are essential for autoinhibition; deletion of either secondary structural element completely relieved EcoRII autoinhibition. This potent regulation principle that keeps EcoRII enzyme activity controlled might protect bacteria against suicidal restriction of rare unmodified recognition sites in the cellular genome.     

Molecular cloning and characterization of ovine homeo-box-containing genes

The sheep genome contains at least eleven homeo-boxes (hox). Using two hox-specific 36-mer oligodeoxynucleotides to screen a sheep genomic library, constructed in lambda Charon28, clones of nine of the hox were identified. Six of the hox clones were analysed by nucleotide sequencing, Southern-blot hybridization and Northern-blot analysis. Two of the hox appear to be cognates of the human Hu-1 (or mouse Hox 2.1) and the mouse Hox 1-3, while another is closely related to the mouse Hox 1-4. These results suggest that there is strong sequence conservation in the hox-containing genes of different mammals, and highlight the possible occurrence of an ubiquitous set of hox-containing genes in mammals. Northern-blot analysis of four sheep hox-containing genes indicates that they are all expressed during embryogenesis and that expression is temporally regulated allowing hierarchical-regulatory interaction. Interestingly, none of the cloned hox-containing sequences contain repetitive sequences.     

Direct monitoring of allosteric recognition of type IIE restriction endonuclease EcoRII

EcoRII is a homodimer with two domains consisting of a DNA-binding N terminus and a catalytic C terminus and recognizes two specific sequences on DNA. It shows a relatively complicated cleavage reaction in bulk solution. After binding to either recognition site, EcoRII cleaves the other recognition site of the same DNA (cis-binding) strand and/or the recognition site of the other DNA (trans-binding) strand. Although it is difficult to separate these two reactions in bulk solution, we could simply obtain the binding and cleavage kinetics of only the cis-binding by following the frequency (mass) changes of a DNA-immobilized quartz-crystal microbalance (QCM) responding to the addition of EcoRII in aqueous solution. We obtained the maximum binding amounts (Deltam(max)), the dissociation constants (K(d)), the binding and dissociation rate constants (k(on) and k(off)), and the catalytic cleavage reaction rate constants (k(cat)) for wild-type EcoRII, the N-terminal-truncated form (EcoRII N-domain), and the mutant derivatives in its C-terminal domain (K263A and R330A). It was determined from the kinetic analyses that the N-domain, which covers the catalytic C-domain in the absence of DNA, preferentially binds to the one DNA recognition site while transforming EcoRII into an active form allosterically, and then the secondary C-domain binds to and cleaves the other recognition site of the DNA strand.     

Molecular cloning of pertussis toxin genes.

We have cloned a 4.5 kb EcoRI/BamHI DNA fragment from Bordetella pertussis which contains at least two genes responsible for expression of pertussis toxin. The S4 subunit of the toxin was isolated by high pressure liquid chromatography and the NH2-terminal amino acid sequence determined. Using a mixed synthetic oligonucleotide probe designed by reverse translation of a portion of the protein sequence, a cloned DNA fragment was identified which contains the coding information for at least the S4 structural subunit of the toxin. Sequence analyses indicate that the mature protein is derived by proteolytic cleavage of a precursor molecule. Southern blot analyses of Tn5-induced B. pertussis toxin-deficient mutants show that the Tn5 DNA is inserted 1.3 kb downstream from the S4 subunit gene. This second gene could code for another subunit required for assembly of the mature toxin or a non-structural transport protein, possibly in the same polycistronic operon. The molecular cloning of pertussis toxin genes provides the basis for development of a safer recombinant "new generation" vaccine for whooping cough.     

Primary sequence of the EcoRII endonuclease and properties of its fusions with beta-galactosidase

The EcoRII endonuclease cleaves DNA containing the sequence CC(A/T)GG before the first cytosine. The methylation of the second cytosine in the sequence by either the EcoRII methylase or Dcm, a chromosomally coded protein in Escherichia coli, inhibits the cleavage. The gene for the EcoRII endonuclease was mapped by analysis of derivatives containing linker insertions, transposon insertions, and restriction fragment deletions. Surprisingly, plasmids carrying the wild-type endonuclease gene and the EcoRII methylase gene interrupted by transposon insertions appeared to be lethal to dcm+ strains of E. coli. We conclude that not all the EcoRII/Dcm recognition sites in the cellular DNA are methylated in dcm+ strains. The DNA sequence of a 1650-base pair fragment containing the endonuclease gene was determined. It revealed an open reading frame that could code for a 45.6-kDa protein. This predicted size is consistent with the known size of the endonuclease monomer (44 kDa). The endonuclease and methylase genes appear to be transcribed convergently from separate promoters. The reading frame of the endonuclease gene was confirmed at three points by generating random protein fusions between the endonuclease and beta-galactosidase, followed by an analysis of the sequence at the junctions. One of these fusions is missing 18 COOH-terminal amino acids of the endonuclease but still displays significant ability to restrict incoming phage in addition to beta-galactosidase activity. No striking similarity between the sequence of the endonuclease and any other protein in the PIR data base was found. The knowledge of the primary sequence of the endonuclease and the availability of the various constructs involving its gene should be helpful in the study of the interaction of the enzyme with its substrate DNA.     

Molecular cloning and DNA homology of plasmid-mediated beta-lactamase genes

Summary Molecular cloning of DNA fragments between 1.5 and 8kb from BamHI, EcoRI, HindIII, SalI, or Sau3A digests permitted the isolation of structural genes coding for TEM-1, ROB-1, OXA-1, OXA-3, OXA-4, OXA-5, PSE-1, PSE-2, PSE-3, PSE-4, CARB-3, CARB-4, AER-1, and LCR-1 -lactamases. Ampicillin-resistant clones were selected and it was confirmed that they contained the respective -lactamase genes by isoelectric focusing. Detailed physical maps of 14 different recombinant plasmids were constructed using 8 restriction endonucleases. Plasmid deletions and lacZ fusions were used to localize the -lactamase structural genes. DNA probes were constructed for the TEM01, ROB-1, OXA-1, and PSE-1 genes. Under conditions of high stringency, hybridization was observed between the genes for TEM-1 and TEM-2 or TLE-1, OXA-1 and OXA-4, and PSE-1 and PSE-4 or CARB-3, while the ROB-1 gene probe showed no cross-hybridization. Such bla gene probes should facilitate studies of -lactamase molecular epidemiology.