科研之友
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摘要
The M . AluI DNA-(cytosine CS)-methyltransferase (5mC methylase) acts on the sequence 5'-AGCT-3'. The amino acid sequences of known 5mC methylases contain ten conserved motifs, with a variable region between Motifs VIII and IX that contains one or more "target-recognizing domains" (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 variable region of all known 5mC methylases, and sequence analysis reveals five candidate TRDs. In testing whether M . AluI is in fact monospecific it was found that AGCT methylation represents only 80-90% of the methylating activity of this enzyme, while control experiments with the enzyme M . HhaI gave no unexplained activity. Because individual TRDs can be deleted from multispecific methylases without general loss of activity, a series of insertion and deletion mutants of the M . AluI variable region were prepared. All deletions that removed more than single amino acids from the variable region caused significant loss of activity; 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 acquiring an additional specificity. When TRDs were moved from a multispecific methylase into two different locations in the variable region of M . AluI, all hybrid enzymes had greatly reduced activity and no new specificities. M . AluI thus behaves in most respects as a monospecific methylase despite the remarkable size of its variable region.
- 出版日期1997-12
