DNA DISTORTION AND BASE FLIPPING BY THE ECORV DNA METHYLTRANSFERASE -A STUDY USING INTERFERENCE AT DA AND T-BASE AND MODIFIED DEOXYNUCLEOSIDE

The EcoRV DNA methyltransferase introduces a CH3 group at the 6-amino position of the first dA in the duplex sequence d(GATATC), It has prev iously been reported that the methylase contacts the four phosphates ( pNpNpGpA) at, and preceding, the 5'-end of the recognition sequence as well as the single dG in this sequence (Szczelkun, M, D,, Jones, H., and Connolly, B, A, (1995) Biochemistry 34, 10734-10743), To study the possible role of the dA and T bases within the ATAT sequence, interfe rence studies have been carried out using diethylpyrocarbonate and osm ium tetroxide, The methylase bound very strongly to hemimethylated oli gonucleotides modified at the second AT, of the ATAT sequence, in the unmethylated strand of the duplex, This probably arises because these modifications facilitate DNA distortion that follows the binding of th e nucleic acid to the protein, Oligonucleotides containing modified ba ses at both the target dA base and its complementary T were used to de termine whether this dA methylase flips out its target base in a simil ar manner to that observed for dC DNA methylases, In binary EcoRV meth ylase-DNA complexes, analogues that weakened the base pair caused an i ncrease in affinity between the protein and the nucleic acid, In contr ast, in ternary EcoRV methylase-DNA-sinefungin (an analogue of the nat ural co factor, S-adenosyl-L-methionine (AdoMet)) complexes, only smal l differences in affinity were observed between the normal dA-T base p air and the analogues, These results are almost identical to those see n with DNA dC methylases (Klimasauskas, S,, and Roberts R, J, (1995) N ucleic Acid Res, 23, 1388-1395; Yang, S, A., Jiang-Cheng, S,, Zingg, J , M,, Mi, S,, and Jones, P, A, (1995) Nucleic Acids Res, 23, 1380-1387 ) and support a base-flipping mechanism for DNA dA methylases.