Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, USA.
J Biol Chem. 2012 Nov 9;287(46):38800-11. doi: 10.1074/jbc.M112.396788. Epub 2012 Sep 13.
The 1,N(6)-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2'-deoxyadenosine (1,N(6)-γ-HMHP-dA) adducts are formed upon bifunctional alkylation of adenine nucleobases in DNA by 1,2,3,4-diepoxybutane, the putative ultimate carcinogenic metabolite of 1,3-butadiene. The presence of a substituted 1,N(6)-propano group on 1,N(6)-γ-HMHP-dA is expected to block the Watson-Crick base pairing of the adducted adenine with thymine, potentially contributing to mutagenesis. In this study, the enzymology of replication past site-specific 1,N(6)-γ-HMHP-dA lesions in the presence of human DNA polymerases (hpols) β, η, κ, and ι and archebacterial polymerase Dpo4 was investigated. Run-on gel analysis with all four dNTPs revealed that hpol η, κ, and Dpo4 were able to copy the modified template. In contrast, hpol ι inserted a single base opposite 1,N(6)-γ-HMHP-dA but was unable to extend beyond the damaged site, and a complete replication block was observed with hpol β. Single nucleotide incorporation experiments indicated that although hpol η, κ, and Dpo4 incorporated the correct nucleotide (dTMP) opposite the lesion, dGMP and dAMP were inserted with a comparable frequency. HPLC-ESI-MS/MS analysis of primer extension products confirmed the ability of bypass polymerases to insert dTMP, dAMP, or dGMP opposite 1,N(6)-γ-HMHP-dA and detected large amounts of -1 and -2 deletion products. Taken together, these results indicate that hpol η and κ enzymes bypass 1,N(6)-γ-HMHP-dA lesions in an error-prone fashion, potentially contributing to A→T and A→C transversions and frameshift mutations observed in cells following treatment with 1,2,3,4-diepoxybutane.
1,N(6)-(2-羟基-3-羟甲基丙-1,3-二基)-2'-脱氧腺苷（1,N(6)-γ-HMHP-dA）加合物是由 1,2,3,4-二环氧丁烷双功能烷基化 DNA 中的腺嘌呤碱基形成的，1,2,3,4-二环氧丁烷是 1,3-丁二烯的潜在致癌代谢物。预计 1,N(6)-γ-HMHP-dA 上取代的 1,N(6)-丙酰基会阻止加合腺嘌呤与胸腺嘧啶的 Watson-Crick 碱基配对，可能导致突变。在这项研究中，研究了在人 DNA 聚合酶（hpols）β、η、κ 和 ι 以及古细菌聚合酶 Dpo4 的存在下，复制特定位置的 1,N(6)-γ-HMHP-dA 损伤的酶学。使用所有四种 dNTP 的运行凝胶分析显示，hpol η、κ 和 Dpo4 能够复制修饰的模板。相比之下，hpol ι 在 1,N(6)-γ-HMHP-dA 对面插入一个碱基，但无法延伸超过受损部位，而 hpol β 观察到完全复制阻断。单核苷酸掺入实验表明，尽管 hpol η、κ 和 Dpo4 在损伤部位对面掺入正确的核苷酸（dTMP），但 dGMP 和 dAMP 的插入频率相当。引物延伸产物的 HPLC-ESI-MS/MS 分析证实了旁路聚合酶在 1,N(6)-γ-HMHP-dA 对面插入 dTMP、dAMP 或 dGMP 的能力，并检测到大量-1 和-2 缺失产物。总之，这些结果表明 hpol η 和 κ 酶以易错的方式绕过 1,N(6)-γ-HMHP-dA 损伤，可能导致在 1,2,3,4-二环氧丁烷处理后细胞中观察到的 A→T 和 A→C 颠换和移码突变。
