Replication Bypass of the <i>trans</i>-4-Hydroxynonenal-Derived (6<i>S</i>,8<i>R</i>,11<i>S</i>)-1,<i>N</i>²-Deoxyguanosine DNA Adduct by the <i>Sulfolobus solfataricus</i> DNA Polymerase IV

Abstract

<i>trans</i>-4-Hydroxynonenal (HNE) is the major peroxidation product of ω-6 polyunsaturated fatty acids in vivo. Michael addition of the <i>N</i>²-amino group of dGuo to HNE followed by ring closure of N1 onto the aldehyde results in four diastereomeric 1,<i>N</i>²-dGuo (1,<i>N</i>²-HNE-dGuo) adducts. The (6<i>S</i>,8<i>R</i>,11<i>S</i>)-HNE-1,<i>N</i>²-dGuo adduct was incorporated into the 18-mer templates 5′-d­(TCAT<u>X</u>GAATCCTTCCCCC)-3′ and d­(TCAC<u>X</u>GAATCCTTCCCCC)-3′, where X = (6<i>S</i>,8<i>R</i>,11<i>S</i>)-HNE-1,<i>N</i>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 (6<i>S</i>,8<i>R</i>,11<i>S</i>)-HNE-1,<i>N</i>²-dGuo adduct, whereas the 14-mer primer allowed analysis of dNTP extension past a primed (6<i>S</i>,8<i>R</i>,11<i>S</i>)-HNE-1,<i>N</i>²-dGuo:dCyd pair. The <i>Sulfolobus solfataricus</i> 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 (6<i>S</i>,8<i>R</i>,11<i>S</i>)-HNE-1,<i>N</i>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 (6<i>S</i>,8<i>R</i>,11<i>S</i>)-HNE-1,<i>N</i>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 (6<i>S</i>,8<i>R</i>,11<i>S</i>)-HNE-1,<i>N</i>²-dGuo adduct, the (6<i>S</i>,8<i>R</i>,11<i>S</i>)-1,<i>N</i>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 (6<i>S</i>,8<i>R</i>,11<i>S</i>)-HNE-1,<i>N</i>²-dGuo:dCyd pair, ring opening of the adduct to the corresponding <i>N</i>²-dGuo aldehyde species occurred. This allowed Watson–Crick base pairing at the (6<i>S</i>,8<i>R</i>,11<i>S</i>)-HNE-1,<i>N</i>²-dGuo:dCyd pair