Replication Bypass of the trans-4-Hydroxynonenal-Derived (6S,8R,11S)-1,N2-Deoxyguanosine DNA Adduct by the Sulfolobus solfataricus DNA Polymerase I

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• 作者：Surajit Banerjee ; Plamen P. Christov ; Albena Kozekova ; Carmelo J. Rizzo ; Martin Egli ; Michael P. Stone
• 刊名：Chemical Research in Toxicology
• 出版年：2012
• 出版时间：February 20, 2012
• 年：2012
• 卷：25
• 期：2
• 页码：422-435
• 全文大小：620K
• 年卷期：v.25,no.2(February 20, 2012)
• ISSN：1520-5010

文摘

trans-4-Hydroxynonenal (HNE) is the major peroxidation product of 蠅-6 polyunsaturated fatty acids in vivo. Michael addition of the N²-amino group of dGuo to HNE followed by ring closure of N1 onto the aldehyde results in four diastereomeric 1,N²-dGuo (1,N²-HNE-dGuo) adducts. The (6S,8R,11S)-HNE-1,N²-dGuo adduct was incorporated into the 18-mer templates 5鈥?d(TCATXGAATCCTTCCCCC)-3鈥?and d(TCACXGAATCCTTCCCCC)-3鈥? where X = (6S,8R,11S)-HNE-1,N²-dGuo adduct. These differed in the identity of the template 5鈥?neighbor base, which was either Thy or Cyt, respectively. Each of these templates was annealed with either a 13-mer primer 5鈥?d(GGGGGAAGGATTC)-3鈥?or a 14-mer primer 5鈥?d(GGGGGAAGGATTCC)-3鈥? The addition of dNTPs to the 13-mer primer allowed analysis of dNTP insertion opposite to the (6S,8R,11S)-HNE-1,N²-dGuo adduct, whereas the 14-mer primer allowed analysis of dNTP extension past a primed (6S,8R,11S)-HNE-1,N²-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,N²-dGuo adduct in a sequence-specific manner. If the template 5鈥?neighbor base was dCyt, the polymerase inserted primarily dGTP, whereas if the template 5鈥?neighbor base was dThy, the polymerase inserted primarily dATP. The latter event would predict low levels of Gua 鈫?Thy mutations during replication bypass when the template 5鈥?neighbor base is dThy. When presented with a primed (6S,8R,11S)-HNE-1,N²-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,N²-dGuo adduct, the (6S,8R,11S)-1,N²-dGuo lesion remained in the ring-closed conformation at the active site. The incoming dNTP, either dGTP or dATP, was positioned with Watson鈥揅rick pairing opposite the template 5鈥?neighbor base, dCyt or dThy, respectively. In contrast, for the 18-mer:14-mer template-primers with a primed (6S,8R,11S)-HNE-1,N²-dGuo:dCyd pair, ring opening of the adduct to the corresponding N²-dGuo aldehyde species occurred. This allowed Watson鈥揅rick base pairing at the (6S,8R,11S)-HNE-1,N²-dGuo:dCyd pair.