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Site-Specific Incorporation of N-(2'-Deoxyguanosine-8-yl)-6-aminochrysene Adduct in DNA and Its Replication in Human Cells.
Chemical Research in Toxicology ( IF 3.7 ) Pub Date : 2020-06-19 , DOI: 10.1021/acs.chemrestox.0c00197 Paritosh Pande 1 , Kimberly R Rebello 1 , Arindom Chatterjee 1 , Spandana Naldiga 1 , Ashis K Basu 1
Chemical Research in Toxicology ( IF 3.7 ) Pub Date : 2020-06-19 , DOI: 10.1021/acs.chemrestox.0c00197 Paritosh Pande 1 , Kimberly R Rebello 1 , Arindom Chatterjee 1 , Spandana Naldiga 1 , Ashis K Basu 1
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The environmental pollutant 6-nitrochrysene (6-NC) is a potent mutagen and a mammary carcinogen in rats. 6-NC is the most potent carcinogen ever tested in the newborn mouse assay. In mammalian cells, it is metabolically activated by nitroreduction and a combination of ring oxidation and nitroreduction pathways. The nitroreduction pathway yields two major adducts with 2′-deoxyguanosine (dG), one at the C8-position, N-(dG-8-yl)-6-AC, and the other at the exocyclic N2-position, 5-(dG-N2-yl)-6-AC. Here, we report the total synthesis of a site-specific oligonucleotide containing the 6-NC-derived C8 dG adduct, N-(dG-8-yl)-6-AC. Pd-catalyzed Buchwald-Hartwig cross coupling of 6-aminochrysene with protected C8-bromo-dG derivative served as the key reaction to furnish protected N-(dG-8-yl)-6-AC in 56% yield. The monomer for solid-phase DNA synthesis was prepared by its deprotection followed by conversion to the corresponding 5′-O-dimethoxytrityl 3′-phosphoramidite, which was used to synthesize a site-specifically adducted oligonucleotide. After purification and characterization, the adduct-containing oligonucleotide was incorporated into a plasmid and replicated in human embryonic kidney (HEK) 293T cells, which showed that N-(dG-8-yl)-6-AC stalls DNA replication as evidenced by 77% translesion synthesis (TLS) efficiency relative to the control and that the adduct is mutagenic (mutation frequency (MF) 17.8%) inducing largely G→T transversions. We also investigated the roles of several translesion synthesis DNA polymerases in the bypass of N-(dG-8-yl)-6-AC using siRNA knockdown approach. TLS efficiency was reduced in hPol η-, hPol κ-, hPol ζ-, and hREV1-deficient HEK 293T cells to 66%, 45%, 37%, and 32%, respectively. Notably, TLS efficiency was reduced to 18% in cells with concurrent knockdown of hPol κ, hPol ζ, and REV1, suggesting that these three polymerases play critical roles in bypassing N-(dG-8-yl)-6-AC. MF increased to 23.1% and 32.2% in hPol κ- and hREV1-deficient cells, whereas it decreased to 11.8% in hPol ζ-deficient cells. This suggests that hPol κ and hREV1 are involved in error-free TLS of this lesion, whereas hPol ζ performs error-prone bypass.
中文翻译：
N-（2'-Deoxyguanosine-8-yl）-6-aminochrysene加合物在DNA中的位点特异性掺入及其在人细胞中的复制。
环境污染物6-硝基丙烯（6-NC）是大鼠的一种强力诱变剂和一种乳致癌物。6-NC是新生小鼠测定中测试过的最有效的致癌物。在哺乳动物细胞中，其通过硝基还原以及环氧化和硝基还原途径的组合被代谢激活。硝基还原途径产生两个带有2'-脱氧鸟苷（dG）的主要加合物，一个在C8位，N-（dG-8-yl）-6-AC，另一个在外环N 2位，5- （dG- N 2-基）-6-AC。在这里，我们报告了包含6-NC衍生的C8 dG加合物N的位点特异性寡核苷酸的总合成-（dG-8-yl）-6-AC。Pd催化6-氨基amino与受保护的C8-溴-dG衍生物的Buchwald-Hartwig交叉偶联是提供受保护的N-（dG-8-基）-6-AC的关键反应，产率为56％。通过脱保护然后转化为相应的5'- O-二甲氧基三苯甲基3'-亚磷酰胺制备用于固相DNA合成的单体，该单体用于合成位点特异性加成的寡核苷酸。纯化和鉴定后，将含加合物的寡核苷酸掺入质粒中，并在人胚肾（HEK）293T细胞中复制，这表明N-（dG-8-yl）-6-AC使DNA复制停顿，这是相对于对照而言77％的跨病变合成（TLS）效率所证明的，并且加合物是诱变的（突变频率（MF）17.8％），主要诱导了G→T颠覆。我们还使用siRNA敲低方法研究了几种跨病变合成DNA聚合酶在N-（dG-8-基）-6-AC旁路中的作用。缺少hPolη-，hPolκ-，hPolζ-和hREV1的HEK 293T细胞的TLS效率分别降低到66％，45％，37％和32％。值得注意的是，在同时敲低hPolκ，hPolζ和REV1的细胞中，TLS效率降低到18％，这表明这三种聚合酶在绕过N方面起关键作用-（dG-8-yl）-6-AC。在缺乏hPolκ和hREV1的细胞中，MF增加到23.1％和32.2％，而在缺乏hPolζ的细胞中，MF降低到11.8％。这表明hPolκ和hREV1参与了该病灶的无错误TLS，而hPolζ执行了容易出错的旁路。
更新日期：2020-07-20
中文翻译：
N-（2'-Deoxyguanosine-8-yl）-6-aminochrysene加合物在DNA中的位点特异性掺入及其在人细胞中的复制。
环境污染物6-硝基丙烯（6-NC）是大鼠的一种强力诱变剂和一种乳致癌物。6-NC是新生小鼠测定中测试过的最有效的致癌物。在哺乳动物细胞中，其通过硝基还原以及环氧化和硝基还原途径的组合被代谢激活。硝基还原途径产生两个带有2'-脱氧鸟苷（dG）的主要加合物，一个在C8位，N-（dG-8-yl）-6-AC，另一个在外环N 2位，5- （dG- N 2-基）-6-AC。在这里，我们报告了包含6-NC衍生的C8 dG加合物N的位点特异性寡核苷酸的总合成-（dG-8-yl）-6-AC。Pd催化6-氨基amino与受保护的C8-溴-dG衍生物的Buchwald-Hartwig交叉偶联是提供受保护的N-（dG-8-基）-6-AC的关键反应，产率为56％。通过脱保护然后转化为相应的5'- O-二甲氧基三苯甲基3'-亚磷酰胺制备用于固相DNA合成的单体，该单体用于合成位点特异性加成的寡核苷酸。纯化和鉴定后，将含加合物的寡核苷酸掺入质粒中，并在人胚肾（HEK）293T细胞中复制，这表明N-（dG-8-yl）-6-AC使DNA复制停顿，这是相对于对照而言77％的跨病变合成（TLS）效率所证明的，并且加合物是诱变的（突变频率（MF）17.8％），主要诱导了G→T颠覆。我们还使用siRNA敲低方法研究了几种跨病变合成DNA聚合酶在N-（dG-8-基）-6-AC旁路中的作用。缺少hPolη-，hPolκ-，hPolζ-和hREV1的HEK 293T细胞的TLS效率分别降低到66％，45％，37％和32％。值得注意的是，在同时敲低hPolκ，hPolζ和REV1的细胞中，TLS效率降低到18％，这表明这三种聚合酶在绕过N方面起关键作用-（dG-8-yl）-6-AC。在缺乏hPolκ和hREV1的细胞中，MF增加到23.1％和32.2％，而在缺乏hPolζ的细胞中，MF降低到11.8％。这表明hPolκ和hREV1参与了该病灶的无错误TLS，而hPolζ执行了容易出错的旁路。
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