Miscodes In Vitro Using Mammalian Polymerases †

8-(hydroxymethyl)-3,N-etheno-dC (8-HM-εC), is a mutagen and animal carcinogen resulting from the reaction of dC with glycidaldehyde. It was synthesized and its phosphoramidite was incorporated site-specifically into a defined 25-mer oligonucleotide. In this study the mutagenic potential of this compound was investigated in an in vitro primer-template extension assay using four mammalian DNA polymerases, and compared to that of the analogous derivative, 3,N-etheno dC (εC). Both adducts primarily blocked replication by calf thymus DNA polymerase α at the modified base, while human polymerase β catalyzed measureable replication synthesis through both adducts. Single nucleotide insertion experiments using pol β showed that dA and dC were incorporated preferentially by both derivatives which resulted in a C T transition or C G transversion. Human polymerase η, a product of the XP-V gene, catalyzed significant bypass of the two lesions, with varying amounts of all four bases incorporated opposite the modified bases. Human polymerase κ primarily blocked synthesis at the base prior to the adduct site. However, some specific misincorporation of dT resulted, forming an εC•T or 8-HM-εC•T pair. From these data, we conclude that the newly synthesized glycidaldehyde-derived adduct, 8-HM-εC, is a miscoding lesion. The similarities in bypass capacity and insertion specificity between the 8-HM-εC and εC bases may be due to the similar planarity and sugar conformations for these two derivatives, as demonstrated by molecular modeling studies.