A GENETIC AND FUNCTIONAL-ANALYSIS OF THE UNUSUALLY LARGE VARIABLE REGION IN THE M-CENTER-DOT-ALUI DNA-(CYTOSINE C5)-METHYLTRANSFERASE

The M . AluI DNA-(cytosine CS)-methyltransferase (5mC methylase) acts on the sequence 5'-AGCT-3'. The amino acid sequences of known 5mC meth ylases contain ten conserved motifs, with a variable region between Mo tifs VIII and IX that contains one or more ''target-recognizing domain s'' (TRDs) responsible for DNA sequence specificity. Monospecific 5mC methylases are believed to have only one TRD, while multispecific 5mC methylases have as many as five. M . AluI has the second-lamest variab le region of all known 5mC methylases, and sequence analysis reveals f ive candidate TRDs. In testing whether M . AluI is in fact monospecifi c it was found that AGCT methylation represents only 80-90% of the met hylating activity of this enzyme, while control experiments with the e nzyme M . HhaI gave no unexplained activity. Because individual TRDs c an be deleted from multispecific methylases without general loss of ac tivity, a series of insertion and deletion mutants of the M . AluI var iable region were prepared. All deletions that removed more than singl e amino acids from the variable region caused significant loss of acti vity; a sensitive in vivo assay for methylase activity based on McrBC restriction suggested that the central portion of the variable region is particularly important. In some cases, multispecific methylases can accommodate a TRD from another multispecific methylase, thereby acqui ring an additional specificity. When TRDs were moved from a multispeci fic methylase into two different locations in the variable region of M . AluI, all hybrid enzymes had greatly reduced activity and no new sp ecificities. M . AluI thus behaves in most respects as a monospecific methylase despite the remarkable size of its variable region.