EVIDENCE FROM INVITRO REPLICATION THAT O6-METHYLGUANINE CAN ADOPT MULTIPLE CONFORMATIONS

The effect of O6-methylguanine (m6G) on replication, in a partially do uble-stranded defined 25-base oligonucleotide, has been studied under nonlimiting conditions of unmodified dNTPs and over an extended time p eriod, using the Klenow fragment of Escherichia coli DNA polymerase I. The sequence surrounding m6G has flanking cytosines (C-m6G-C), and th e initial steady-state kinetics have been reported. When the primer wa s annealed so that the first base to be replicated was m6G, replicatio n was virtually complete in almost-equal-to 5 min, although the reacti on appears biphasic. When annealed with a primer where thymine or cyto sine is paired opposite template m6G, about half the molecules were re plicated in the first 15 sec, and no significant further replication w as seen over a 1-hr period. When m6G was dealkylated by DNA-O6-methylg uanine-methyltransferase, replication was rapid with no blockage. Thes e data suggest that there can be two (or more) conformations of m6G. I n these studies the term syn refers to conformers interfering with bas e-pairing, whereas anti refers to those allowing such base-pairing. Pr evious physical studies by others indicate that syn- and anti-conforme rs of the methyl group relative to the N1 of guanine are possible. Her e molecular modeling/computational studies are described, suggesting t hat syn- and anti-m6G can be of similar energy in DNA, and, therefore, these two conformers may explain the two types of species observed du ring in vitro replication. An alternative explanation could be the pos sibility that the different species may manifest differential interact ions of m6G with Klenow fragment. These results may provide a rational e for why m6G lesions in vivo have been reported to be lethal as well as mutagenic.