8-(Hydroxymethyl)-3,N4-etheno-C (8-HM-εC) is an exocyclic adduct resulting from the reaction of dC with glycidaldehyde, a mutagen and animal carcinogen. This compound has now been synthesized and its phosphoramidite incorporated site-specifically into a defined 25-mer oligonucleotide. In this study, the mutagenic potential of this adduct in the 25-mer oligonucleotide was investigated in an in vitro primertemplate extension assay using four mammalian DNA polymerases. The miscoding potentials were also compared to those of an analogous derivative, 3,N4-etheno C (εC), in the same sequence. Both adducts primarily blocked replication by calf thymus DNA polymerase α at the modified base, while human polymerase β catalyzed measurable replication synthesis through both adducts. Nucleotide insertion experiments showed that dA and dC were incorporated by pol β opposite either adduct, which would result in a C → T transition or C → G transversion. Human polymerase η, a product of the xeroderma pigmentosum variant (XP-V) gene, catalyzed the most efficient bypass of the two lesions with 25% and 32% for 8-HM-εC and εC bypassed after 15 min. Varying amounts of all four bases opposite the modified bases resulted with pol η. Human polymerase κ primarily blocked synthesis at the base prior to the adduct. However, some specific misincorporation of dT resulted, forming an εC·T or 8-HM-EC·T pair. From these data, we conclude that the newly synthesized glycidaldehyde-derived adduct, 8-HM-εC, is a miscoding lesion. The bypass efficiency and insertion specificity of 8-HM-εC and εC were similar for all four polymerases tested, which could be attributed to the similar planarity and sugar conformations for these two derivatives as demonstrated by molecular modeling studies.
|Number of pages||8|
|State||Published - Feb 12 2002|
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