Replication Bypass of the trans-4-Hydroxynonenal-Derived (6S,8R,11S)-1,N2-Deoxyguanosine DNA Adduct by the Sulfolobus solfataricus DNA Polymerase IV
Replication Bypass of the trans-4-Hydroxynonenal-Derived (6S,8R,11S)-1,N2-Deoxyguanosine DNA Adduct by the Sulfolobus solfataricus DNA Polymerase IV
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DOI：
10.1021/tx200460j
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发表时间：
2012-02-01
影响因子：
4.1
通讯作者：
Stone, Michael P.
中科院分区：
文献类型：
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作者：
Banerjee, Surajit;Christov, Plamen P.;Stone, Michael P.
反式-4-羟基壬烯醛（HNE）是ω-6多不饱和脂肪酸在体内的主要过氧化产物。将dGuo的N-2-氨基迈克尔加成到HNE上，然后将N1闭环到醛上，得到四种非对映体1，N-2-dGuo（1，N-2-HNE-dGuo）加合物。将（6S，8 R，11 S）-HNE-1，N-2-dGuo加合物引入到18聚体模板5 ′-d（TCATXGAATCCTTCCCCC）-3 ′和d（TCACXGAATCCTTCCCCC）-3 ′中，其中X =（6S，8 R，11 S）-HNE-1，N-2-dGuo加合物。这些不同之处在于模板5 '-相邻碱基的身份，其分别是Thy或Cyt。将这些模板中的每一个与13-mer引物5 '-d（GGGGGAAGGATTC）-3'或14-mer引物5 '-d（GGGGGAAGGATTCC）-3'退火。将dNTP添加到13-mer引物允许分析与（6S，8 R，11 S）-HNE-1，N-2-dGuo加合物相对的dNTP插入，而14-mer引物允许分析dNTP延伸通过引发的（6S，8 R，11 S）-HNE-1，N-2-dGuo：dCyd对。硫磺硫化叶菌P2 DNA聚合酶IV（Dpo 4）属于易错聚合酶的Y家族。体外复制旁路研究表明，该聚合酶以序列特异性方式插入dNTPs与（6S，8 R，11 S）-HNE-1，N-2-dGuo加合物相对。如果模板5 '-相邻碱基是dCyt，则聚合酶主要插入dGTP，而如果模板5'-相邻碱基是dThy，则聚合酶主要插入dATP。当模板5 '-相邻碱基是dThy时，后一事件将预测复制旁路期间低水平的Gua -> Thy突变。当呈现引发的（6S，8 R，11 S）-HNE-1，N-2-dGuo：dCyd对时，聚合酶进行全长引物延伸。获得了具有所有四种模板-引物的三元（Dpo 4-DNA-dNTP）复合物的结构。对于其中聚合酶与（6S，8 R，11 S）-HNE-1，N-2-dGuo加合物面对的18-mer：13-mer模板-引物，（6S，8 R，11 S）-1，N-2-dGuo损伤在活性位点保持闭环构象。将引入的dNTP（dGTP或dATP）分别与模板5 '-相邻碱基dCyt或dThy相对地用沃森克里克配对定位。相比之下，对于具有引发的（6S，8 R，11 S）-HNE-1，N-2-dGuo：dCyd对的18-mer：14-mer模板-引物，发生加合物开环为相应的N-2-dGuo醛物质。这允许在（6S，8 R，11 S）-HNE-1，N-2-dGuo：dCyd对处的沃森克里克碱基配对。
trans-4-Hydroxynonenal (HNE) is the major peroxidation product of omega-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.