The Small Subunit of M · Aqu I Is Responsible for Sequence-Specific DNA Recognition and Binding in the Absence of the Catalytic Domain

Abstract

ABSTRACT Aqu I DNA methyltransferase (M ·  Aqu I) 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 ·  Aqu I is a heterodimer in which the polypeptide chain is separated at the junction between the two equivalent structural domains in the related enzyme M ·  Hha I. Recently, we reported the subcloning, overexpression, and purification of the subunits (α and β) of M ·  Aqu I separately. Here we describe the DNA binding properties of M ·  Aqu I. The results presented here indicate that the β 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 ·  Aqu I 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 β subunit. The α subunit enhances the binding of the β subunit to DNA specifically and nonspecifically. It is likely that the addition of the α subunit to the β subunit stabilizes the conformation of the β 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 α subunit. These compounds did not have any effect on DNA binding by the β subunit alone. Using a 30-mer oligodeoxynucleotide substrate containing 5-fluorodeoxycytidine (5-FdC), it was found that the β 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 α subunit to the β subunit led to the formation of a covalent complex with specific DNA sequence containing 5-FdC.