SEQUENCE-SPECIFIC BINDING OF DNA BY THE ECORV RESTRICTION AND MODIFICATION ENZYMES WITH NUCLEIC-ACID AND COFACTOR ANALOGS

The DNA-binding properties of the EcoRV restriction endonuclease and m odification methyltransferase with their recognition sequence (GATATC) were analyzed using the electrophoretic band-shift assay. It has prev iously been observed that the endonuclease does not bind specifically to GATATC sequences in the absence of the essential cofactor Mg2+. To investigate any possible roles for Mg2+ in promoting specific DNA bind ing, a set of hydrolysis-resistant oligonucleotide substrates were syn thesized that contained either phosphate (phosphorothioate, 3'-S-phosp horothiolate), sugar (4'-thiothymidine), or base (7-deaza-2'-deoxyaden osine) modifications. However, it was found that none of these were sp ecifically bound by the endonuclease in either the absence or the pres ence of Mg2+. In contrast, the methylase bound to GATATC sequences muc h more strongly than to nonspecific sites, and it was possible to obse rve the formation of enzyme-DNA complexes by gel retardation. Binding to GATATC sequences was increased by the addition of sinefungin, a non reactive analogue of the essential cofactor S-adenosyl-L-methionine (A doMet). Presumably this also occurs with AdoMet although methylation a nd turnover prevented its direct observation. In the presence of sinef ungin the strongest binding was observed with hemimethylated EcoRV seq uences (K-d = 11-13 nM), and unmethylated DNA was bound less well (K-d = 46 nM). Specific, albeit weaker binding was also seen with the dime thylated product (K-d = 143 nM). A difference in electrophoretic mobil ity was observed between enzyme-substrate and enzyme-product complexes suggestive of structural differences between them. The K-app value fo und for sinefungin, with the hemimethylated EcoRV sequence, was 10.9 m M.