SPECIFICITIES OF HUMAN, RAT AND ESCHERICHIA-COLI O-6-METHYLGUANINE-DNA METHYLTRANSFERASES TOWARDS THE REPAIR OF O-6-METHYL AND O-6-ETHYLGUANINE IN DNA

The behaviour of highly purified bacterial expressed rat O-6-methylguanine-DNA methyltransferase (MGMT) towards the repair of CGCm6GAGCTCGCG and CGCe6GAGCTCGCG (k(m6G)/k(e6G) = 1.45, where k is the second order repair rate constant determined, m6G and e6G are O-6-methyl and O-6-ethylguanine) is similar to that of E.coli 39kD Ada protein (k(m6G)/k(e6G) = 1.6). However, the human MGMT is very different (k(m6G)/k(e6G) = 163). The preferential repair of O-6-ethylguanine lesion by the rat MGMT appears not to be related to the lack of the initiator methionine in the expressed protein since similar results were obtained from N-terminal Glutathione-S-transferase (GST) fused protein (GSTMGMT) which retains the methionine. The possible relationship between these findings and the differences observed in the primary amino acid sequence of these proteins is discussed. In addition the preferential repair of O-6-ethylguanine substrate by the 39kD Ada protein as compared to the catalytic C-terminus alone (different by 134 times) suggests that the N-terminus plays a crucial role in the repair of O-6-ethylguanine. This is in constrast to the minor effects of the GST domain when fused to the N-terminus of mammalian MGMT.