New bifunctional restriction-modification enzyme AloI isoschizomer (PcoI): Bioinformatics analysis, purification and activity confirmation.

Type II restriction endonucleases and modification DNA-methyltransferases are key instruments of genetic engineering. Recently the number of proteins assigned to this group exceeds 8500. Subtype IIC organizes bifunctional endonuclease-methyltransferase enzymes and currently consists of 16 described members.

Binding of DNA methyltransferase M.Ecl18kI [corrected] to operator-promoter region decreases its methylating activity.

The type II bifunctional DNA methyltransferase (MTase) Ecl18 that is able to control transcription of its own gene was studied kinetically. Based on initial velocity dependences from S-adenosyl-L-methionine (AdoMet) and target DNA and substrate preincubation assays, it is proposed that the enzyme apparently works by a rapid equilibrium ordered bi-bi mechanism with DNA binding first. By measuring the enzyme activity depending on DNA and AdoMet at different fixed concentrations of the operator sequence oligonucleotide, it was found that its binding has noncompetitive inhibitory effect on Ecl18 MTase activity.

Fused eco29kIR- and M genes coding for a fully functional hybrid polypeptide as a model of molecular evolution of restriction-modification systems.

Background: The discovery of restriction endonucleases and modification DNA methyltransferases, key instruments of genetic engineering, opened a new era of molecular biology through development of the recombinant DNA technology. Today, the number of potential proteins assigned to type II restriction enzymes alone is beyond 6000, which probably reflects the high diversity of evolutionary pathways. Here we present experimental evidence that a new type IIC restriction and modification enzymes carrying both activities in a single polypeptide could result from fusion of the appropriate genes from preexisting bipartite restriction-modification systems.

6His-Eco29kI methyltransferase methylation site and kinetic mechanism characterization.

A new type II 6His-Eco29kI DNA methyltransferase was tested for methylation site (CC(Me)GCGG) and catalytic reaction optimal conditions. With high substrate concentrations, an inhibitory effect of DNA, but not AdoMet, on its activity was observed. Isotope partitioning and substrate preincubation assays showed that the enzyme-AdoMet complex is catalytically active.

Construction of an overproducing strain, purification, and biochemical characterization of the 6His-Eco29kI restriction endonuclease.

We constructed a strain of Escherichia coli overproducing 6His-tagged Eco29kI by placing the coding sequence under control of a strong bacteriophage T5 promoter. The yield of 6His-Eco29kI restriction endonuclease expression could be increased to about 20% of the total cellular protein, but inclusion bodies formed consisting of insoluble 6His-Eco29kI protein. We developed a fast and effective protocol for purification of the homogeneous enzyme from both soluble and insoluble fractions and established their identity by catalytic activity assay.