ABSTRACT
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. Here we present phylogenetic tree of 22 new potential bifunctional endonucleases. The majority of them are thought to be fusions of a restriction nuclease with a DNA-methyltransferase and a target recognition subunit of type I restriction-modification systems (R-M-S structure). A RM.AloI isoschizomer from Prevotella copri DSM-18205, PcoI, has been cloned, purified and its REase activity demonstrated. It cuts DNA in magnesium-dependent manner and demonstrates high affinity to DNA, which probably reflects its mechanism of action. This work provides additional proves that gene fusion might play an important role in evolution of restriction-modification systems and other DNA-modifying proteins.
Subject(s)
Bacterial Proteins/chemistry , DNA Modification Methylases/chemistry , Deoxyribonucleases, Type II Site-Specific/chemistry , Prevotella/enzymologyABSTRACT
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. RESULTS: Fusion of eco29kIR and M ORFs gave a novel gene encoding for a fully functional hybrid polypeptide that carried both restriction endonuclease and DNA methyltransferase activities. It has been placed into a subclass of type II restriction and modification enzymes--type IIC. Its MTase activity, 80% that of the M.Eco29kI enzyme, remained almost unchanged, while its REase activity decreased by three times, concurrently with changed reaction optima, which presumably can be caused by increased steric hindrance in interaction with the substrate. In vitro the enzyme preferentially cuts DNA, with only a low level of DNA modification detected. In vivo new RMS can provide a 102-fold less protection of host cells against phage invasion. CONCLUSIONS: We propose a molecular mechanism of appearing of type IIC restriction-modification and M.SsoII-related enzymes, as well as other multifunctional proteins. As shown, gene fusion could play an important role in evolution of restriction-modification systems and be responsible for the enzyme subclass interconversion. Based on the proposed approach, hundreds of new type IIC enzymes can be generated using head-to-tail oriented type I, II, and III restriction and modification genes. These bifunctional polypeptides can serve a basis for enzymes with altered recognition specificities. Lastly, this study demonstrates that protein fusion may change biochemical properties of the involved enzymes, thus giving a starting point for their further evolutionary divergence.
Subject(s)
Bacterial Proteins/metabolism , Biological Evolution , DNA Modification Methylases/metabolism , Deoxyribonucleases, Type II Site-Specific/metabolism , Peptides/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , DNA Modification Methylases/genetics , DNA Restriction-Modification Enzymes/chemistry , DNA Restriction-Modification Enzymes/genetics , DNA Restriction-Modification Enzymes/metabolism , Deoxyribonucleases, Type II Site-Specific/chemistry , Deoxyribonucleases, Type II Site-Specific/genetics , Molecular Sequence Data , Peptides/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence AlignmentABSTRACT
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. Considering effect of different concentrations of DNA and AdoMet on initial velocity, ping-pong mechanisms were ruled out. According to data obtained, the enzyme appears to work by preferred ordered bi-bi mechanism with AdoMet as leading substrate.
Subject(s)
DNA Methylation , DNA-Cytosine Methylases/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/enzymology , Adenosine/analogs & derivatives , Adenosine/chemistry , Binding Sites , Catalysis , Ethionine/analogs & derivatives , Ethionine/chemistry , Kinetics , Substrate SpecificityABSTRACT
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. The isolated enzymes were tested for recognition specificity and optimal reaction conditions as a function of NaCl and KCl concentrations, temperature, and pH compared with the native Eco29kI restriction endonuclease. The 6His-tagged enzyme retained the specificity of the native protein but had an altered optimum of its catalytic reaction.
