EXACT SIZE AND ORGANIZATION OF DNA TARGET-RECOGNIZING DOMAINS OF MULTISPECIFIC DNA-(CYTOSINE-C5)-METHYLTRANSFERASES

A large portion of the sequences of type II DNA(cytosine-C5)-methyltra nsferases (C5-MTases) represent highly conserved blocks of amino acids , General steps in the methylation reaction performed by C5-MTases hav e 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 capac ity of each C5-MTase to recognize its characteristic target. Individua l 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 identi fied in the C-terminal part of the variable region of multispecific C5 -MTases. With experiments reported here, we have established the organ ization of the variable regions of the multispecific MTases M.SPRI, M. phi 3TI, M.H2I and M.rho 11(s)I at the resolution of individual amino acids. These regions comprise 204, 175, 268 and 268 amino acids, respe ctively, All variable regions are bipartite. They contain at their N-t erminal 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 stud ied, 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 e nzymes with equal specificity have the same size, TRDs do not overlap but are either separated by linker amino acids or abut each other.