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 subunits, BcgI A and BcgI B. Both are required for endonuclease and methyltransferase activities. Here, we show that the endonuclease activity is mediated by the N-terminal portion of the A subunit. We made this assignment by mutational analysis. The analytic strategy involved three steps. First, the methyltransferase activity was inactivated by site-directed mutagenesis of a conserved methyltransferase motif also found in the A subunit. One of the R+M- mutants could not methylate DNA but was still able to cleave it, therefore expression of this mutant gene was lethal to the host. This lethal phenotype allowed the selective isolation of cleavage-deficient (R-) mutations in a second round of random mutagenesis in this mutant background. The R- mutations were all located in the N-terminal portion of the A subunit. There are five potential endonuclease motifs within this region. Conserved acidic residues in 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. Further support for this assignment was obtained by another round of site-directed mutagenesis directed to residues surrounding this motif. The results showed that DNA cleavage activity was mediated by the predicted, conserved residues, and not any of the surrounding non-conserved residues. One mutant protein, BcgI-E53A, with a single amino acid substitution decreased the DNA cleavage activity at least 700-fold. Our present model for the functional organization of BcgI locates both endonuclease and methyltransferase domains in the A subunit, with the target recognition domain located in the B subunit.
Kong
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New England Biolabs, 32 Tozer Road, Beverly, MA, 01915, USA.
D000595:Amino Acid Sequence; D001483:Base Sequence; D015280:DNA Restriction-Modification Enzymes; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D016415:Sequence Alignment
D015280:DNA Restriction-Modification Enzymes; C073736:endodeoxyribonuclease BcgI; D015252:Deoxyribonucleases, Type II Site-Specific