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 meth yl group at the 6-NH2 position of the first deoxyadenosine in GATATC s equences. The enzyme forms a stable and specific complex with GATATC s equences in the presence of a nonreactive analogue, such as sinefungin , of its natural cofactor S-adenosyl-L-methionine. Using circular perm utation band mobility shift analysis (in which the distance between th e GATATC sequence and the end of the DNA is varied) of protein-DNA-cof actor complexes we have shown the methylase induces a bend of just ove r 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 th e induced curve was toward the major groove. There was no significant difference in the bend angle measured using unmethylated GATATC sequen ces and hemimethylated sequences which contain G(6-Me)ATATC in one str and only. These are the natural substrates for the enzyme. The EcoRV e ndonuclease, a very well characterized protein, served as a positive c ontrol. DNA bending by this protein has been previously determined bot h 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 degrees, is a little larger than that observe d with the endonuclease, which is approximately 44 degrees.