CLONING AND ANALYSIS OF THE 4 GENES-CODING FOR BPU10I RESTRICTION-MODIFICATION ENZYMES

The Bpu10I R-M system from Bacillus pumilus 10, which recognizes the a symmetric 5'-CCTNAGC sequence, has been cloned, sequenced and expresse d in Escherichia coli, The system comprises four adjacent, similarly o riented genes encoding two m5C MTases and two subunits of Bpu10I ENase (34.5 and 34 kDa), Both bpu10IR genes either in cis or trans are need ed for the manifestation of R.Bpu10I activity. Subunits of R.Bpu10I, p urified to apparent homogeneity, are both required for cleavage activi ty, This heterosubunit structure distinguishes the Bpu10I restriction endonuclease from all other type II restriction enzymes described prev iously, The subunits reveal 25% amino acid identity, Significant simil arity was also identified between a 43 amino acid region of R.DdeI and one of the regions of higher identity shared between the Bpu10I subun its, a region that could possibly include the catalytic/Mg2+ binding c enter, The similarity between Bpu10I and DdeI MTases is not limited to the conserved motifs (CM) typical for m5C MTases, It extends into the variable region that lies between CMs VIII and IX, Duplication of a p rogenitor gene, encoding an enzyme recognizing a symmetric nucleotide sequence, followed by concerted divergent evolution, may provide a pos sible scenario leading to the emergence of the Bpu10I ENase, which rec ognizes an overall asymmetric sequence and cleaves within it symmetric ally.