Molecular enzymology of the EcoRV DNA-(Adenine-N (6))-methyltransferase: kinetics of DNA binding and bending, kinetic mechanism and linear diffusion of the enzyme on DNA

Abstract

The EcoRV DNA-(adenine-N(6))-methyltransferase recognizes GATATC sequences and modifies the first adenine residue within this site. We show here, that the enzyme binds to the DNA and the cofactor S-adenosylmethionine (AdoMet) in an ordered bi-bi fashion, with AdoMet being bound first. M.EcoRV binds DNA in a non-specific manner and the enzyme searches for its recognition site by linear diffusion with a range of approximately 1800 bp. During linear diffusion the enzyme continuously scans the DNA for the presence of recognition sites. Upon specific M.EcoRV-DNA complex formation a strong increase in the fluorescence of an oligonucleotide containing a 2-aminopurine base analogue at the GAT-2AP-TC position is observed which, most likely, is correlated with DNA bending. In contrast to the GAT-2AP-TC substrate, a G-2AP-TATC substrate in which the target base is replaced by 2-aminopurine does not show an increase in fluorescence upon M.EcoRV binding, demonstrating that 2-aminopurine is not a general tool to detect base flipping. Stopped-flow experiments show that DNA bending is a fast process with rate constants >10 s(-1). In the presence of cofactor, the specific complex adopts a second conformation, in which the target sequence is more tightly contacted by the enzyme. M.EcoRV exists in an open and in a closed state that are in slow equilibrium. Closing the open state is a slow process (rate constant approximately 0.7 min(-1)) that limits the rate of DNA methylation under single turnover conditions. Product release requires opening of the closed complex which is very slow (rate constant approximately 0.05-0.1 min(-1)) and limits the rate of DNA methylation under multiple turnover conditions. M.EcoRV methylates DNA sequences containing more than one recognition sites in a distributive manner. Since the dissociation rate from non-specific DNA does not depend on the length of the DNA fragment, DNA dissociation does not preferentially occur at the ends of the DNA.