Cloning of NruI and Sbo13I restriction and modification sstems in E. coli and amino acid sequence comparison of M.NruI and M.Sbo13I with other amino-methyltransferases.

BACKGROUND

NruI and Sbo13I are restriction enzyme isoschizomers with the same recognition sequence 5' TCG downward arrowCGA 3' (cleavage as indicated downward arrow). Here we report the cloning of NruI and Sbo13I restriction-modification (R-M) systems in E. coli. The NruI restriction endonuclease gene (nruIR) was cloned by PCR and inverse PCR using primers designed from the N-terminal amino acid sequence. The NruI methylase gene (nruIM) was derived by inverse PCR walking.

RESULTS

The amino acid sequences of NruI endonuclease and methylase are very similar to the Sbo13I R-M system which has been cloned and expressed in E. coli by phage selection of a plasmid DNA library. Dot blot analysis using rabbit polyclonal antibodies to N6mA- or N4mC-modified DNA indicated that M.NruI is possibly a N6mA-type amino-methyltransferase that most likely modifies the external A in the 5' TCGCGA 3' sequence. M.Sbo13I, however, is implicated as a probable N4mC-type methylase since plasmid carrying sbo13IM gene is not restricted by Mrr endonuclease and Sbo13I digestion is not blocked by Dam methylation of the overlapping site. The amino acid sequence of M.NruI and M.Sbo13I did not show significant sequence similarity to many known amino-methyltransferases in the alpha, beta, and gamma groups, except to a few putative methylases in sequenced microbial genomes.

CONCLUSIONS

The order of the conserved amino acid motifs (blocks) in M.NruI/M.Sbo13I is similar to the gamma. group amino-methyltranferases, but with two distinct features: In motif IV, the sequence is DPPY instead of NPPY; there are two additional conserved motifs, IVa and Xa as extension of motifs IV and X, in this family of enzymes. We propose that M.NruI and M.Sbo13I form a subgroup in the gamma group of amino-methyltransferases.