Cytosine C5-methylation plays important roles in several biological phenomena, such as restriction-modification in prokaryotes, genomic imprinting and carcinogenesis in eukaryotes [1
]. This reaction is catalyzed by DNA (cytosine-5) methyltransferases (C5-MTase), which transfer a methyl group from S-adenosyl-methionine (AdoMet) to carbon 5 of cytosine in specific DNA sequences [2
]. Prokaryotic C5-MTases contain ten conserved sequence motifs and a so-called variable region located between conserved motifs VIII and IX [3
]. The conserved motifs are responsible for the general steps of the methyl transfer reaction [4
], whereas specific sequence recognition is mediated mainly by the variable region [10
]. Our understanding of the three-dimensional structure of C5-MTases and of their interaction with substrate DNA is mainly based on the X-ray structures of two enzymes: M.HhaI and M.HaeIII. They revealed that both MTases fold in two domains, the large domain encompassing most of the conserved motifs and the small domain containing the variable region and conserved motif IX. The two domains form a cleft where the DNA substrate fits with the major groove facing the small domain and the minor groove facing the large domain. All specific DNA-protein interactions are at the small domain – major groove interface [12
]. Eukaryotic C5-MTases are larger proteins but the sequence homology they share with prokaryotic C5-MTases and the available biochemical data suggest that they have the same catalytic mechanism [14
Although the vast majority of characterized C5-MTases function as monomers, there are exceptions: M.AquI (C
YCGRG) and M.EcoHK31I (YGGC
CR) consist of two polypeptides. The larger subunit of M.AquI contains conserved motifs I – VIII and part of the variable region, whereas the smaller subunit contains the distal half of the variable region and conserved motifs IX - X [16
]. In M.EcoHK31I, the larger subunit encompasses conserved motifs I – VIII, X as well as the predicted target recognition domain (TRD), and only motif IX is located in the smaller polypeptide [18
]. The structural plasticity of C5-MTases is also supported by the phenomenon of protein fragment complementation observed with three enzymes: N- and C-terminal inactive fragments of three naturally monomeric C5-MTases (M.BspRI, M.BsuRI and M.HhaI) can assemble to form active MTase if expressed in the same E. coli
The goal of this work was to test whether M.SssI, which has the same specificity (CG) as the eukaryotic DNA MTases [21
] and therefore has special importance as an experimental tool in the study of eukaryotic DNA methylation, is capable of fragment complementation. In higher eukaryotes, DNA methylation occurs at CG dinucleotides (CpG sites) and is associated with gene silencing [22
]. Targeted DNA methylation, i.e. selective methylation of predetermined CpG sites in the genome is emerging as a promising technique for selective gene silencing [23
]. The applicability of targeted methylation as a research tool or as a potential therapeutic approach critically depends on the specificity of targeting, i. e.
on the difference of methylation between targeted and non-targeted sites. One approach to increase targeting specificity capitalized on the phenomenon of functional complementation between inactive fragments of the MTase. In the first implementation of this technique complementing N- and C-terminal fragments of the HhaI MTase were genetically fused to zinc finger proteins (ZFP) engineered to recognize different nine bp sequences. When the MTase fragment-ZFP fusion proteins were expressed in the same E. coli
cell, the targeted M.HhaI recognition site, which was flanked by the two closely spaced ZFP binding sites, became methylated, whereas the other M.HhaI recognition sites on the same plasmid stayed unmethylated [28
]. Although this strategy is likely to require improvement to suppress the non-targeted background methylation mainly deriving from the reconstitution of the MTase in unbound state [29
], it probably remains the most promising approach for achieving the specificity required for using targeted methylation as a reliable research tool [30
]. However, of the C5-MTases shown to possess the capacity of fragment complementation, only M.HhaI can be used to target CpG sites, and even M.HhaI can methylate only a small subset of CpG sites (1 in 16, those in GC
GC context). To be able to target any CpG site, one needs a C5-MTase with CG specificity such as M.SssI.
Here, we report that M.SssI shows the phenomenon of fragment complementation, thus it is, in principle, suitable for the split fragment approach of targeted DNA methylation.