ANALYZING THE FUNCTIONAL-ORGANIZATION OF A NOVEL RESTRICTION-MODIFICATION SYSTEM, THE BCGI SYSTEM

BcgI is a novel, multi-subunit, restriction-modification (R-M) system that differs from all the other types of R-M system in its genetic and functional organization. The holoenzyme contains two different subuni ts, BcgI A and BcgI B. Both are required for endonuclease and methyltr ansferase activities. Here, we show that the endonuclease activity is mediated by the N-terminal portion of the A subunit. We made this assi gnment by mutational analysis. The analytic strategy involved three st eps. First, the methyltransferase activity was inactivated by site-dir ected mutagenesis of a conserved methyltransferase motif also found in the A subunit. One of the R+M- mutants could not methylate DNA but wa s still able to cleave it, therefore expression of this mutant gene wa s lethal to the host. This lethal phenotype allowed the selective isol ation of cleavage-deficient (R-) mutations in a second round of random mutagenesis in this mutant background. The R- mutations were all loca ted in the N-terminal portion of the A subunit. There are five potenti al endonuclease motifs within this region. Conserved acidic residues i n each of these motifs were substituted with alanine by site-directed mutagenesis of the wild-type A gene. The results identified one motif, P52E53-(X)(12)-E66D67K68, as the probable endonuclease active-site. F urther support for this assignment was obtained by another round of si te-directed mutagenesis directed to residues surrounding this motif. T he results showed that DNA cleavage activity was mediated by the predi cted, conserved residues, and not any of the surrounding non-conserved residues. One mutant protein, BcgI-E53A, with a single amino acid sub stitution decreased the DNA cleavage activity at least 700-fold. Our p resent model for the functional organization of BcgI locates both endo nuclease and methyltransferase domains in the A subunit, with the targ et recognition domain located in the B subunit. (C) 1998 Academic Pres s Limited.