Effect of sequence context on O-6-methylguanine repair and replication in vivo

Understanding the origins of mutational hotspots is complicated by the inte rtwining of several variables. The selective formation, repair, and replica tion of a DNA lesion, such as O-6-methylguanine (m(6)G), can, in principle, be influenced by the surrounding nucleotide environment. A nearest-neighbo r analysis was used to address the contribution of sequence context on m(6) G repair by the Escherichia coli methyltransferases Ada or Ogt, and on DNA polymerase infidelity in vivo. Sixteen M13 viral genomes with m6G flanked b y all permutations of G, A, T, and C were constructed and individually tran sformed into repair-deficient and repair-proficient isogenic cell strains. The 16 genomes were introduced in duplicate into 5 different cellular backg rounds for a total of 160 independent experiments, for which mutations were scored using a recently developed assay. The Ada methyltransferase demonst rated strong 5' and 3' sequence-specific repair of m(6)G in vivo. The Ada 5 ' preference decreased in the general order: GXN > CXN > TXN > AXN (X = m(6 )G, N = any base), while the Ada 3' preference decreased in the order: NX(T /C) > NX(G/A), with mutation frequencies (MFs) ranging from 35% to 90%. The Ogt methyltransferase provided MFs ranging from 10% to 25%. As was demonst rated by Ada, the Ogt methyltransferase repaired m(6)G poorly in an AXN con text. When both methyltransferases were removed, the MF was nearly 100% for all sequence contexts, consistent with the view that the replicative DNA p olymerase places T opposite m(6)G during replication irrespective of the lo cal sequence environment.