The EcoRV modification methylase causes considerable bending of DNA upon binding to its recognition sequence GATATC

The EcoRV methyltransferase modifies DNA by the introduction of a methyl group at the 6-NH2 position of the first deoxyadenosine in GATATC sequences. The enzyme forms a stable and specific complex with GATATC sequences in the presence of a nonreactive analogue, such as sinefungin, of its natural cofactor S-adenosyl-L-methionine. Using circular permutation band mobility shift analysis (in which the distance between the GATATC sequence and the end of the DNA is varied) of protein-DNA-cofactor complexes we have shown the methylase induces a bend of just over 60 degrees in the bound DNA. This was confirmed by phasing analysis, in which the spacing between the GATATC site and a poly(dA) tract is varied through a helical turn, which showed that the orientation of the induced curve was toward the major groove. There was no significant difference in the bend angle measured using unmethylated GATATC sequences and hemimethylated sequences which contain G6-Me ATATC in one strand only. These are the natural substates for the enzyme. The EcoRV endonuclease, a very well characterized protein, served as a positive control. DNA bending by this protein has been previously determined both by crystallographic and solution methods. The two proteins bend DNA toward the major groove but the bend angle produced by the methylase, slightly greater than 60 degree, is a little larger than that observed with the endonuclease, which is approximately 44 degrees.