The small subunit of M. AquI is responsible for sequence-specific DNA recognition and binding in the absence of the catalytic domain.

Abstract

AquI DNA methyltransferase (M. AquI) catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to the C5 position of the outermost deoxycytidine base in the DNA sequence 5'-CCCGGG-3'. M. AquI is a heterodimer in which the polypeptide chain is separated at the junction between the two equivalent structural domains in the related enzyme M. HhaI. Recently, we reported the subcloning, overexpression, and purification of the subunits (alpha and beta) of M. AquI separately. Here we describe the DNA binding properties of M. AquI. The results presented here indicate that the beta subunit alone contains all of the information for sequence-specific DNA recognition and binding. The first step in the sequence-specific recognition of DNA by M. AquI involves the formation of binary complex with the target recognition domain in conjunction with conserved sequence motifs IX and X, found in all known C5 DNA methyltransferases, contained in the beta subunit. The alpha subunit enhances the binding of the beta subunit to DNA specifically and nonspecifically. It is likely that the addition of the alpha subunit to the beta subunit stabilizes the conformation of the beta subunit and thereby enhances its affinity for DNA indirectly. Addition of S-adenosyl-L-methionine and its analogues S-adenosyl-L-homocysteine and sinefungin enhances binding, but only in the presence of the alpha subunit. These compounds did not have any effect on DNA binding by the beta subunit alone. Using a 30-mer oligodeoxynucleotide substrate containing 5-fluorodeoxycytidine (5-FdC), it was found that the beta subunit alone did not form a covalent complex with its specific sequence in the absence or presence of S-adenosyl-L-methionine. However, the addition of the alpha subunit to the beta subunit led to the formation of a covalent complex with specific DNA sequence containing 5-FdC.