Exact size and organization of DNA target-recognizing domains of multispecific DNA-(cytosine-C5)-methyltransferases.

Abstract

A large portion of the sequences of type II DNA‐(cytosine‐C5)‐methyltransferases (C5‐MTases) represent highly conserved blocks of amino acids. General steps in the methylation reaction performed by C5‐MTases have been found to be mediated by some of these domains. C5‐MTases carry, in addition at the same relative location, a region variable in size and amino acid composition, part of which is associated with the capacity of each C5‐MTase to recognize its characteristic target. Individual target‐recognizing domains (TRDs) for the targets CCGG (M), CC(A/T)GG (E), GGCC (H), GCNGC (F) and G(G/A/T)GC(C/A/T)C (B) could be identified in the C‐terminal part of the variable region of multispecific C5‐MTases. With experiments reported here, we have established the organization of the variable regions of the multispecific MTases M.SPRI, M.phi3TI, M.H2I and M.rho 11SI at the resolution of individual amino acids. These regions comprise 204, 175, 268 and 268 amino acids, respectively. All variable regions are bipartite. They contain at their N‐terminal side a very similar sequence of 71 amino acids. The integrity of this sequence must be assured to provide enzyme activity. Bracketed by 6–10 ‘linker’ amino acids, they have, depending on the enzyme studied, towards their C‐terminal end ensembles of individual TRDs of 38 (M), 39 (E), 40 (H), 44 (F) and 54 (B) amino acids. TRDs of different enzymes with equal specificity have the same size. TRDs do not overlap but are either separated by linker amino acids or abut each other.

Removal of a frameshift between the hsdM and hsdS genes of the EcoKI Type IA DNA restriction and modification system produces a new type of system and links the different families of Type I systems

“…Such an assembly would have an ‘extra’ copy of the HsdS subunit and this presumably would have to be pushed out of the way when assembling the RM enzyme. The presence of multiple TRDs, some of which must be pushed out of the way when assembling an active enzyme, is not without precedent as some H. pylori Type I RM systems appear to have three TRDs in their HsdS (32) and some Type II MTases contain as many as five, consecutive, functional TRDs (though only one TRD is active at a time) (51). However given the results of the western blot (Figure 5), we believe that the presence of some HsdM is the most likely reason for activity.…”

Genetic variation in ICF syndrome: Evidence for genetic heterogeneity

“…The catalytic domain contains the highly conserved methyltransferase motifs I, IV, VI, IX and X [Xie et al, 1999]; the similarity with motif VIII is much weaker [Hansen et al, 1999]. The motif VIII to IX region is known to be important for interaction with DNA in many methyltransferases and is the major determinant of target site specificity for monospecific and multispecific m5 C-methyltransferases [Trautner et al, 1996]. The mutations, V726G and V818M, have also been found in ICF patients Cu and M, respectively (indicated by #) [Xu et al, 1999].…”

The DNMT3B DNA methyltransferase gene is mutated in the ICF immunodeficiency syndrome

“…The V726G mutation occurs in a region with weak similarity to motif VIII, and the in-frame SerThrPro insertion occurs in the middle of motif IX. The motif VIII to IX region is known to be important for interaction with DNA in many methyltransferases and is the major determinant of target site specificity for monospecific and multispecific m5 C-methyltransferases (27).…”