Monika Reuter

Oligonucleotide duplexes containing CC(A/T)GG stimulate cleavage of refractory DNA by restriction endonuclease EcoRII

Some DNA species are resistant towards the restriction endonuclease EcoRII despite the presence of unmodified recognition sites. We show that 14 base‐pair oligonucleotide duplexes containing the EcoRII recognition site 5′‐CC(A/T)GG are cleaved by this enzyme and are able to stimulate EcoRII cleavage of such resistant DNA molecules (e.g. DNA of bacterial virus T3). A direct correlation between the concentration of oligonucleotide duplex molecules and the degree of EcoRII digestion of the primarily resistant DNA is observed. This indicates a stoichiometric rather than a catalytic mode of enzyme activation. An excess of DNA devoid of EcoRII sites (‘non‐site’ DNA, e.g. MvaI‐digested T7 DNA) does not interfere with the activity of EcoRII.

Avoidance of DNA methylation. A virus-encoded methylase inhibitor and evidence for counterselection of methylase recognition sites in viral genomes.

Theocr+ gene of bacterial virus T7 codes for the first protein recognized to inhibit a specific group of DNA methylases. The recognition sequences of several other DNA methylases, not susceptible to Ocr inhibition, are significantly suppressed in the virus genome. The bacterial virus T3 encodes an Ado-Met hydrolase, destroying the methyl donor and causing T3 DNA to be totally unmethylated.These observations could stimulate analogous investigations into the regulation of DNA methylation patterns of eukaryotic viruses and cells. For instance, an underrepresentation of methylation sites (5′-CG) is also true for animal DNA viruses.Moreover, we were able to disclose some novel properties of DNA restriction-modification enzymes concerning the protection of DNA recognition sequences in which only one strand can be methylated (e.g., type III enzymeEcoP15) and the primary resistance of (unmethylated) DNA recognition sites towards type II restriction endonucleaseEcoRII.

Restriction endonucleases functionally interacting with two DNA sites

Simultaneous interaction with two recognition sites was found to be a precondition for DNA cleavage by certain type-II and type-III restriction endonucleases. Nevertheless, the molecular mechanisms of the protein-DNA interaction are different between members of both classes of enzymes.

An improved method for the detection of Dcm methylation in DNA molecules

The intrinsic insensitivity of EcoRII recognition sites in RF DNAs of phage M13 and vector M13mp18 towards this restriction endonuclease can be overcome by adding site-specific oligodeoxyribonucleotide duplexes to the restriction sample. Since Dcm- DNA but not Dcm(+)-methylated DNA becomes susceptible under these conditions, this procedure constitutes an improvement of the Dcm methylation assay.

A new crystal form of restriction endonuclease EcoRII that diffracts to 2.8 ˚ A resolution

EcoRII, a type IIe restriction endonuclease, has been crystallized in space group P2(1), with unit-cell parameters a = 58.3, b = 127.8, c = 59.9 A, beta = 91.4 degrees. There are two monomers in the asymmetric unit and the solvent content is estimated to be 49% by volume. The crystals diffract to 2.8 A resolution, which is much higher than that of the previously reported cubic crystal form, which diffracted to 4 A resolution.

Overproduction of His-tagged EcoRII restriction endonuclease and terminally deleted mutant proteins ☆

EcoRII was the first restriction endonuclease (ENase) reported requiring the cooperative interaction with at least two DNA sites for activity. Using two different expression systems the enzyme could be purified and its special substrate requirements were further analyzed. At the present state of knowledge we suggest a model of simultaneous binding of two DNA sites to one dimeric enzyme molecule (see Krüger et al., FEMS Microbiol. Rev. (1995) in press).

Structure and Function of Type IIE Restriction Endonucleases — or: From a Plasmid That Restricts Phage Replication to A New Molecular DNA Recognition Mechanism

Restriction endonucleases (REases) and DNA methyltransferases (MTases) are encoded by chromosomal, plasmid, or viral genes. In eubacteria as well as in archaea, they often form biologically active DNA restriction-modification (RM) systems (for a review see Kruger and Reuter 1999).The first R-M systems, discovered by reversible growth reduction of bacterial viruses, were found to be encoded by chromosomal genes in Escherichia coli K-12, Escherichia coli B, and Salmonella typhimurium (for reviews: Arber 1974; Boyer 1971; Meselson et al. 1972). Besides the prophage PI and the related plasmid p15 (Glover et al. 1963; Arber and Wauters-Willems 1970), also naturally occurring drug resistance plasmids “R factors”) of the fi: (fertility inhibition-minus) type have been shown to restrict and modify infecting bacteriophage or trans-conjugated plasmid DNAs in vivo. The first R-factor controlled R-M systems, called EcoRI and EcoRII today, have been defined by those phenotypic properties (Arber and Morse 1965; Bannister and Glover 1968, 1970; Watanabe et al. 1964). Endonucleolysis (Takano et al. 1968; Yoshimori et al.1972) and cytosine methylation (Hattman 1972), respectively, have been proposed as the molecular mechanisms of EcoRII-specific restriction and modification. EcoRII was among the very first R-M enzymes for which the DNA substrate site was identified (Bigger et al. 1973; Boyer et al. 1973). The REase EcoRII recognizes the sequence 5’-CC(AlT)GG which exhibits a twofold rotational symmetry with a dyad axis corresponding to the central A-T pair.