title: Fused eco29kIR- and M genes coding for a fully functional hybrid polypeptide as a model of molecular evolution of restriction-modification systems.
abstract: 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.
authors.affiliation: Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Prospekt Nauki, 5 Pushchino, Moscow region 142290, Russia.
authors.forename: Marina L
authors.initials: ML
authors.lastname: Mokrishcheva
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Solonin
authors.forename: Dmitri V
authors.initials: DV
authors.lastname: Nikitin
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005075:Biological Evolution; D015254:DNA Modification Methylases / Q000235:genetics / Q000378:metabolism*; D015280:DNA Restriction-Modification Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D015280:DNA Restriction-Modification Enzymes; D010455:Peptides; D011993:Recombinant Fusion Proteins; D015254:DNA Modification Methylases; C035337:CCGCGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific