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.

Figure 1

Overproducing strain construction and purification of protein RM.Eco29kI. A - eco29kIR and M ORFs orientation on the natural plasmid pECO29 [5]. On the plasmid Stop codon of eco29kIR gene overlaps Start codon of eco29kIM gene. B - A scheme of site-directed mutagenesis used for generation of the fusion protein RM.Eco29kI. Overlapped Stop and Start codons were substitutes for two Glycine codons. C - 10% PAGE electrophoresis with RM.Eco29kI induction (lane 2); final preparation of the enzyme (lane 3). Lane 1 - protein extract from non-induced cells, M - Dalton markers of 26, 34, 47, 86 and 120 kDA.

Figure 2

Agarose gel visualization of the phage φ80vir DNA hydrolysis pattern with RM.Eco29kI (lane 3) and R.Eco29kI (lane 4). Lane 1 - Dalton markers: 1000, 1500, 2000, 2500, 3000, 4000, 5000, 6000, 8000, and 10000 bp; lane 2 - uncut phage φ80vir DNA.

Figure 3

Reaction optima characterization for RM.Eco29kI. Triangles above gels mark 2-fold dilutions of the enzyme in the reaction mixtures. First lane of each gel represents uncut phage φ80vir DNA. The gels represent RM.Eco29kI activity depending on pH, temperature, NaCl and KCl concentrations. Varied parameters are written above each gel.

Figure 4

Reaction optima characterization for RM.Eco29kI, continuation of Figure 3.

Figure 5

Phage restrictions by RM.Eco29k1. Triangle marks phage dilutions which are shown above. BL21(DE3)xp29k11 cells carry only gene coding for Eco29kI MTase and lack Eco29kI REase activity. BL21(DE3)xpECO29 cells carry natural pECO29 plasmid, having both MTase and REase activities of the wild type Eco29kI RMS. BL21(DE3)xp29k11+p29RM cells carry genes coding for M.Eco29kI and RM.Eco29kI enzymes on two different plasmids.

Figure 6

Analysis of the AdoMet effect on REase activity of RM.Eco29kI. A - reactions were carried out in REase optimal buffer in the absence (left panel, -AdoMet) and presence (right panel, +AdoMet) of AdoMet; B - reactions were carried out in MTase optimal buffer in the absence (left panel, -AdoMet) and presence (right panel, +AdoMet) of AdoMet. Triangles above gels mark 2-fold dilutions of the enzyme in the reaction mixtures. The figure represents ratio of REase and MTase activities in RM.Eco29kI enzyme.

Figure 7

Organization schemes of proved type IIC RMS. Black arrows show localization of corresponding ORFs. R' - REase part; M' - MTase part; S' - DNA recognition parts of enzymes. The figure demonstrates gene organization of characterized type IIC RMS.

Figure 8

Organization schemes of proved type IIC RMS, continuation of Figure 7.

Figure 9

A scheme of genetic rearrangement events that could lead to single ORF fusions. Grey and black arrows show any hypothetical ORFs; white circle arrow - inversion of DNA; triangles - recombination between different pieces of DNA. On the figure are shown genetic pathways that could lead to gene fusion.

Figure 10

A scheme of alignment for M.SsoII and M.NlaX. Identical amino acids are marked by black and similar amino acids by grey background. The figure demonstrates presence of 70 aa N-terminal domain in M.SsoII, but not in M.NlaX.