Replication bypass of the trans-4-hydroxynonenal-derived (6s,8R,11S)-1,N 2-deoxyguanosine DNA adduct by the sulfolobus solfataricus DNA polymerase IV

Abstract

Trans-4-Hydroxynonenal (HNE) is the major peroxidation product of ω-6 polyunsaturated fatty acids in vivo. Michael addition of the N 2-amino group of dGuo to HNE followed by ring closure of N1 onto the aldehyde results in four diastereomeric 1,N 2-dGuo (1,N 2-HNE-dGuo) adducts. The (6S,8R,11S)-HNE-1,N 2-dGuo adduct was incorporated into the 18-mer templates 5′-d(TCATXGAATCCTTCCCCC)- 3′ and d(TCACXGAATCCTTCCCCC)-3′, where X = (6S,8R,11S)-HNE-1,N 2-dGuo adduct. These differed in the identity of the template 5′-neighbor base, which was either Thy or Cyt, respectively. Each of these templates was annealed with either a 13-mer primer 5′-d(GGGGGAAGGATTC)- 3′ or a 14-mer primer 5′-d(GGGGGAAGGATTCC)-3′. The addition of dNTPs to the 13-mer primer allowed analysis of dNTP insertion opposite to the (6S,8R,11S)-HNE-1,N 2-dGuo adduct, whereas the 14-mer primer allowed analysis of dNTP extension past a primed (6S,8R,11S)-HNE-1,N 2-dGuo: dCyd pair. The Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) belongs to the Y-family of error-prone polymerases. Replication bypass studies in vitro reveal that this polymerase inserted dNTPs opposite the (6S,8R,11S)-HNE-1,N 2-dGuo adduct in a sequence-specific manner. If the template 5′-neighbor base was dCyt, the polymerase inserted primarily dGTP, whereas if the template 5′-neighbor base was dThy, the polymerase inserted primarily dATP. The latter event would predict low levels of Gua → Thy mutations during replication bypass when the template 5′-neighbor base is dThy. When presented with a primed (6S,8R,11S)-HNE-1,N 2-dGuo:dCyd pair, the polymerase conducted full-length primer extension. Structures for ternary (Dpo4-DNA-dNTP) complexes with all four template-primers were obtained. For the 18-mer:13-mer template-primers in which the polymerase was confronted with the (6S,8R,11S)-HNE-1,N 2-dGuo adduct, the (6S,8R,11S)-1,N 2-dGuo lesion remained in the ring-closed conformation at the active site. The incoming dNTP, either dGTP or dATP, was positioned with Watson-Crick pairing opposite the template 5′-neighbor base, dCyt or dThy, respectively. In contrast, for the 18-mer:14-mer template-primers with a primed (6S,8R,11S)-HNE-1,N 2-dGuo:dCyd pair, ring opening of the adduct to the corresponding N 2-dGuo aldehyde species occurred. This allowed Watson-Crick base pairing at the (6S,8R,11S)-HNE-1,N 2-dGuo:dCyd pair. © 2012 American Chemical Society.