Translesion synthesis across 1,N2-ethenoguanine by human DNA polymerases

Abstract

1,N2-Etheno(ε)guanine (ε) is formed in DNA as a result of exposure to certain vinyl monomers (e.g., vinyl chloride) or from lipid peroxidation. This lesion has been shown to be mutagenic in bacteria and mammalian cells. 1,N2-ε-G has been shown to block several model replicative DNA polymerases (pols), with limited bypass. Recently, an archebacterial DNA pol, Sulfolobus solfataricus Dpo4, has been shown to copy past 1,N2-ε-G. In this study, we examined the abilities of recombinant, full-length human pol δ and three human translesion DNA pols to copy past 1,N2-ε-G. The replicative pol, pol δ, was completely blocked. Pols ι and κ showed similar rates of incorporation of dTTP and dCTP. Pol η was clearly the most active of these pols in copying past 1,N2-ε-G, incorporating in the order dGTP > dATP > dCTP, regardless of whether the base 5′ of 1,N 2-ε-G in the template was C or T. Pol η also had the highest error frequency opposite 1,N2-ε-G. Analysis of the extended products of the pol η reactions by mass spectrometry indicated only two products, both of which had G incorporated opposite 1,N2-ε-G and all other base pairing being normal (i.e., G:C and A:T). One-half of the products contained an additional A at the 3′-end, presumably arising from a noninformational blunt end addition or possibly a slipped insertion mechanism at the end of the primer-template replication process. In summary, the most efficient of the four human DNA pols was pol η, which appeared to insert G opposite 1,N2-ε-G and then copy correctly. This pattern differs with the same oligonucleotide sequences and 1,N2-ε-G observed using Dpo4, emphasizing the importance of pols in mutagenesis events. © 2006 American Chemical Society.