[Anti-Restriction Activity of ArdB Protein against EcoAI Endonuclease].

ArdB proteins are known to inhibit the activity of the type I restriction-modification (RM-I) system, in particular EcoKI (IA family). The mechanism of ArdB's activity still remains unknown; the spectrum of targets inhibited has been poorly studied. In this work, it was shown that the presence of the ardB gene from the R64 plasmid could suppress the activity of EcoAI endonuclease (IB family) in Escherichia coli TG1 cells.

Crystal structure of a novel domain of the motor subunit of the Type I restriction enzyme EcoR124 involved in complex assembly and DNA binding.

Although EcoR124 is one of the better-studied Type I restriction-modification enzymes, it still presents many challenges to detailed analyses because of its structural and functional complexity and missing structural information. In all available structures of its motor subunit HsdR, responsible for DNA translocation and cleavage, a large part of the HsdR C terminus remains unresolved. The crystal structure of the C terminus of HsdR, obtained with a crystallization chaperone in the form of pHluorin fusion and refined to 2.

A residue of motif III positions the helicase domains of motor subunit HsdR in restriction-modification enzyme EcoR124I.

Type I restriction-modification enzymes differ significantly from the type II enzymes commonly used as molecular biology reagents. On hemi-methylated DNAs type I enzymes like the EcoR124I restriction-modification complex act as conventional adenine methylases at their specific target sequences, but unmethylated targets induce them to translocate thousands of base pairs through the stationary enzyme before cleaving distant sites nonspecifically. EcoR124I is a superfamily 2 DEAD-box helicase like eukaryotic double-strand DNA translocase Rad54, with two RecA-like helicase domains and seven characteristic sequence motifs that are implicated in translocation.

The helical domain of the EcoR124I motor subunit participates in ATPase activity and dsDNA translocation.

Type I restriction-modification enzymes are multisubunit, multifunctional molecular machines that recognize specific DNA target sequences, and their multisubunit organization underlies their multifunctionality. EcoR124I is the archetype of Type I restriction-modification family IC and is composed of three subunit types: HsdS, HsdM, and HsdR. DNA cleavage and ATP-dependent DNA translocation activities are housed in the distinct domains of the endonuclease/motor subunit HsdR.

Functional coupling of duplex translocation to DNA cleavage in a type I restriction enzyme.

Type I restriction-modification enzymes are multifunctional heteromeric complexes with DNA cleavage and ATP-dependent DNA translocation activities located on motor subunit HsdR. Functional coupling of DNA cleavage and translocation is a hallmark of the Type I restriction systems that is consistent with their proposed role in horizontal gene transfer. DNA cleavage occurs at nonspecific sites distant from the cognate recognition sequence, apparently triggered by stalled translocation.

Interdomain communication in the endonuclease/motor subunit of type I restriction-modification enzyme EcoR124I.

Restriction-modification systems protect bacteria from foreign DNA. Type I restriction-modification enzymes are multifunctional heteromeric complexes with DNA-cleavage and ATP-dependent DNA translocation activities located on endonuclease/motor subunit HsdR. The recent structure of the first intact motor subunit of the type I restriction enzyme from plasmid EcoR124I suggested a mechanism by which stalled translocation triggers DNA cleavage via a lysine residue on the endonuclease domain that contacts ATP bound between the two helicase domains.

Antibacterial, cytotoxicity and physical properties of laser--silver doped hydroxyapatite layers.

Hydroxyapatite layers with silver doping from 0.06 at.% to 14 at.

The interrelationship of helicase and nuclease domains during DNA translocation by the molecular motor EcoR124I.

The type I restriction-modification enzyme EcoR124I comprises three subunits with the stoichiometry HsdR2/HsdM2/HsdS1. The HsdR subunits are archetypical examples of the fusion between nuclease and helicase domains into a single polypeptide, a linkage that is found in a great many other DNA processing enzymes. To explore the interrelationship between these physically linked domains, we examined the DNA translocation properties of EcoR124I complexes in which the HsdR subunits had been mutated in the RecB-like nuclease motif II or III.

Characterization of a restriction modification system from the commensal Escherichia coli strain A0 34/86 (O83:K24:H31).

Background: Type I restriction-modification (R-M) systems are the most complex restriction enzymes discovered to date. Recent years have witnessed a renaissance of interest in R-M enzymes Type I. The massive ongoing sequencing programmes leading to discovery of, so far, more than 1 000 putative enzymes in a broad range of microorganisms including pathogenic bacteria, revealed that these enzymes are widely represented in nature.

A RecB-family nuclease motif in the Type I restriction endonuclease EcoR124I.

The Type I restriction-modification enzyme EcoR124I is an ATP-dependent endonuclease that uses dsDNA translocation to locate and cleave distant non-specific DNA sites. Bioinformatic analysis of the HsdR subunits of EcoR124I and related Type I enzymes showed that in addition to the principal PD-(E/D)xK Motifs, I, II and III, a QxxxY motif is also present that is characteristic of RecB-family nucleases. The QxxxY motif resides immediately C-terminal to Motif III within a region of predicted alpha-helix.

Phosphorylation of Type IA restriction-modification complex enzyme EcoKI on the HsdR subunit.

Phosphorylation of Type I restriction-modification (R-M) enzymes EcoKI, EcoAI, and EcoR124I - representatives of IA, IB, and IC families, respectively - was analysed in vivo by immunoblotting of endogenous phosphoproteins isolated from Escherichia coli strains harbouring the corresponding hsd genes, and in vitro by a phosphorylation assay using protein kinase present in subcellular fractions of E. coli. From all three R-M enzymes, the HsdR subunit of EcoKI system was the only subunit that was phosphorylated.

Structural model for the multisubunit Type IC restriction-modification DNA methyltransferase M.EcoR124I in complex with DNA.

Recent publication of crystal structures for the putative DNA-binding subunits (HsdS) of the functionally uncharacterized Type I restriction-modification (R-M) enzymes MjaXIP and MgeORF438 have provided a convenient structural template for analysis of the more extensively characterized members of this interesting family of multisubunit molecular motors. Here, we present a structural model of the Type IC M.EcoR124I DNA methyltransferase (MTase), comprising the HsdS subunit, two HsdM subunits, the cofactor AdoMet and the substrate DNA molecule.

Post-translational modification(s) and cell distribution of Streptomyces aureofaciens translation elongation factor Tu overproduced in Escherichia coli.

We cloned EF-Tu from Streptomyces aureofaciens on a pET plasmid and overproduced it using the T7 RNA polymerase system in Escherichia coli. Streptomyces EF-Tu represented more than 40% of the total cell protein and was stored mostly in inclusion bodies formed apically at both ends of E. coli cells.

Cellular localization of Type I restriction-modification enzymes is family dependent.

Cellular localization of Type I restriction-modification enzymes EcoKI, EcoAI, and EcoR124I-the most frequently studied representatives of IA, IB, and IC families-was analyzed by immunoblotting of subcellular fractions isolated from Escherichia coli strains harboring the corresponding hsd genes. EcoR124I shows characteristics similar to those of EcoKI. The complex enzymes are associated with the cytoplasmic membrane via DNA interaction as documented by the release of the Hsd subunits from the membrane into the soluble fraction following benzonase treatment.

Identification of the EcoKI and EcoR124I Type I restriction--modification enzyme subunits by non-equilibrium pH gradient two-dimensional gel electrophoresis.

Effectively optimized and reproducible procedure for monitoring the composition of type I restriction-modification endonucleases EcoKI and EcoR124I by non-equilibrium pH gradient two-dimensional (2-D) gel electrophoresis is described. Three subunits of the enzyme complex, which widely differ from one another in their isoelectric points and molar mass, were identified in crude cell extracts of E. coli.

A novel mutant of the type I restriction-modification enzyme EcoR124I is altered at a key stage of the subunit assembly pathway.

The HsdS subunit of a type I restriction-modification (R-M) system plays an essential role in the activity of both the modification methylase and the restriction endonuclease. This subunit is responsible for DNA binding, but also contains conserved amino acid sequences responsible for protein-protein interactions. The most important protein-protein interactions are those between the HsdS subunit and the HsdM (methylation) subunit that result in assembly of an independent methylase (MTase) of stoichiometry M(2)S(1).

Localization of the type I restriction-modification enzyme EcoKI in the bacterial cell.

To localise the type I restriction-modification (R-M) enzyme EcoKI within the bacterial cell, the Hsd subunits present in subcellular fractions were analysed using immunoblotting techniques. The endonuclease (ENase) as well as the methylase (MTase) were found to be associated with the cytoplasmic membrane. HsdR and HsdM subunits produced individually were soluble, cytoplasmic polypeptides and only became membrane-associated when coproduced with the insoluble HsdS subunit.

Two temperature-sensitive mutations in the DNA binding subunit of EcoKI with differing properties.

Two temperature-sensitive mutations in the hsdS gene, which encodes the DNA specificity subunit of the type IA restriction-modification system EcoKI, designated Sts1 (Ser(340)Phe) and Sts2 (Ala(204)Thr) had a different impact on restriction-modification functions in vitro and in vivo. The enzyme activities of the Sts1 mutant were temperature-sensitive in vitro and were reduced even at 30 degrees C (permissive temperature). Gel retardation assays revealed that the Sts1 mutant had significantly decreased DNA binding, which was temperature-sensitive.

The effect of recA mutation on the expression of EcoKI and EcoR124I hsd genes cloned in a multicopy plasmid.

Type I restriction-modification (R-M) endonucleases are composed of three subunits--HsdR, required for restriction, and HsdM and HsdS which can produce a separate DNA methyltransferase. The HsdS subunit is required for DNA recognition. In this paper we describe the effect of cloned EcoKI and EcoR124I hsd genes on the resulting R-M phenotype.

Isolation of a non-classical mutant of the DNA recognition subunit of the type I restriction endonuclease R.EcoR124I.

We have used deletion mutagenesis and PCR-based misincorporation mutagenesis to produce a collection of mutations in the central conserved region of the DNA binding subunit of the type IC restriction endonuclease EcoR124I. It has been proposed that this domain is involved in protein-protein interactions during the assembly of the endonuclease. While a large percentage of these mutations gave a classical Res- Mod- phenotype, one mutant was isolated with a nonclassical Res- Mod+ phenotype.

Overproduction of the Hsd subunits leads to the loss of temperature-sensitive restriction and modification phenotype.

The genes hsdM and hsdS for M. EcoKI modification methyltransferase and the complete set of hsdR, hsdM and hsdS genes coding for R. EcoKI restriction endonuclease, both with and without a temperature-sensitive (ts) mutation in hsdS gene, were cloned in pBR322 plasmid and introduced into E.

Restriction endonucleases R.EcoKI and R.EcoR124I are probably located in different environments within the bacterial cell.

We describe the phenomenon of a transient state of R124I restriction deficiency after long-term storage of the E. coli[pCP1005] strain at 4 degrees C, or after growth of the culture in synthetic M9 medium with the nonmutagenic solvent dimethyl sulfoxide. The unusual high reversion from the R+ 124 to the R- 124 phenotype was observed only in E.

Cloning, production and characterisation of wild type and mutant forms of the R.EcoK endonucleases.

The hsdR, hsdM and hsdS genes coding for R.EcoK restriction endonuclease, both with and without a temperature sensitive mutation (ts-1) in the hsdS gene, were cloned in pBR322 plasmid and introduced into E.coli C3-6.

Mutation in the specificity polypeptide of the type I restriction endonuclease R.EcoK that affects subunit assembly.

We describe the isolation and characterization of a temperature-sensitive mutation within the hsdS gene of the type I restriction and modification system EcoK. This mutation appears to affect the ability of the HsdR subunit to interact with the HsdS subunit when forming an active endonuclease. We discuss the possibility that this mutant, together with another mutation described previously, may define a discontinuous domain, involved in protein-protein interactions, within the HsdS polypeptide.

A mutation that converts serine340 of the HsdSK polypeptide to phenylalanine and its effects on restriction and modification in Escherichia coli K-12.

A hybrid hsdS gene, encoding the HsdSts + d polypeptide, was constructed by joining the proximal region of the wild-type (wt) hsdS sequence with the distal region of the hsdSts + d sequence, at the hsdS BglII site. The hybrid hsdS-Sts + d gene exerts a trans-dominant effect on restriction and modification, which points to the location of the temperature-sensitive (ts) trans-dominant (+ d) mutation in the gene hsdSts + d distal region. Sequencing of the region downstream from the HindIII target in the Escherichia coli K-12 hsdSts + d mutant was carried out.