Why do O6-alkylguanine and O4-alkylthymine miscode? The relationship between the structure of DNA containing O6-alkylguanine and O4-alkylthymine and the mutagenic properties of these bases.

The carcinogenic and mutagenic N-nitroso compounds produce GC to AT and TA to GC transition mutations because they alkylate O6 of guanine and O4 of thymine. It has been generally assumed that these mutations occur because O6-alkylguanine forms a stable mispair with thymine and O4-alkylthymine forms a mispair with guanine. Recent studies have shown that this view is mistaken and that the alkylG.T and alkylT.G mispairs are not more stable than their alkylG.C or alkylT.A counterparts. Two possible explanations based on recent structural studies are put forward to account for the miscoding. The first possibility is that the DNA polymerase might mistake O6-alkylguanine for adenine, and O4-alkylthymine for cytosine, because of the physical similarity of these bases. O6-Methylguanine and adenine are similarly lipophilic and X-ray crystallography of the nucleosides has shown a close similarity in bond angles and lengths between O6-methylguanine and adenine, and between O4-methylthymine and cytosine. The second possible explanation is that the important factor in the miscoding is that the alkylG.T and alkylT.G mispairs retain the Watson-Crick alignment with N1 of the purine juxtaposed to N3 of the pyrimidine while the alkylG.C and alkylT.A pairs adopt a wobble conformation. 31P NMR of DNA duplexes show that the phosphodiester links both 3' and 5' to the C have to be distorted to accommodate the O6-ethylguanine:C pair, whereas there is less distortion of the phosphodiesters 3' and 5' to the T in an ethylG.T pair. Recent kinetic measurements show that the essential aspect of base selection in DNA synthesis is the ease of formation of the phosphodiester links on both the 3' and 5' side of the incoming base. The Watson-Crick alignment of the alkylG.T and alkylT.G mispairs may facilitate formation of these phosphodiester links, and this alignment rather than the strength of the base pairs and the extent of hydrogen bonding between them may be the crucial factor in the miscoding. If either hypothesis is correct it suggests that previously too much emphasis has been placed on the stability of the normal pairs in the replication of DNA.