Langouët S, Müller M, Guengerich F P
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Biochemistry. 1997 May 20;36(20):6069-79. doi: 10.1021/bi962526v.
1,N2-Ethenoguanine (1,N2-epsilon-Gua) and 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine (HO-ethanoGua) are two modified bases formed in the reaction of DNA with 2-chlorooxirane, the epoxide derivative of vinyl chloride. The oligonucleotides (19-mers), 5'-CAGTGGGTGTCCGAATTGA-3', were prepared, with each of these modified bases substituted for G at G. HO-ethanodeoxyguanosine exists predominantly as a mixture of diastereomers of the closed cyclic hemiaminal form, 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine, shown by H2(18)O experiments to be in equilibrium with the open form, N2-(2-oxoethyl)Gua. Both adducts retarded the 3'-extension of a complementary 10-mer primer by all of the polymerases examined, but in every case, some full-length product was obtained. Nucleotide sequence analysis indicated misincorporation of dGTP and dATP across from both 1,N2-epsilon-Gua and HO-ethanoGua, with the extent varying considerably among the polymerases. Similar results were obtained when the abilities of the polymerases to incorporate a single dNTP were evaluated. In addition, -1 and -2 base frame shifts were detected with both 1,N2-epsilon-Gua and HO-ethanoGua with some of the polymerases. Steady-state kinetic experiments with Escherichia coli polymerase I exo- and T7 polymerase exo-/thioredoxin showed large decreases in k(cat) for all dNTP incorporations compared to the normal G x dCTP pair and high misincorporation frequencies for dATP and dGTP with both adducts (compared to dCTP). Collectively, the results indicate that both of these adducts have considerable miscoding potential with some of these polymerases, that there are a number of differences between the 1,N2-epsilon-Gua and HO-ethanoGua adducts (which formally differ only in the presence of the elements of water), and that misincorporation of dNTPs at a single modified base can vary considerably among different polymerases even in the absence of exonuclease activity.
1,N2-乙烯基鸟嘌呤（1,N2-ε-鸟嘌呤）和5,6,7,9-四氢-7-羟基-9-氧代咪唑并[1,2-a]嘌呤（HO-乙醇基鸟嘌呤）是DNA与2-氯环氧乙烷（氯乙烯的环氧化物衍生物）反应形成的两种修饰碱基。制备了寡核苷酸（19聚体）5'-CAGTGGGTGTCCGAATTGA-3'，其中这些修饰碱基中的每一个都替代了G处的G。HO-乙醇基脱氧鸟苷主要以闭环半缩醛形式的非对映异构体混合物存在，即5,6,7,9-四氢-7-羟基-9-氧代咪唑并[1,2-a]嘌呤，H2(18)O实验表明其与开放形式N2-(2-氧代乙基)鸟嘌呤处于平衡状态。两种加合物均使所检测的所有聚合酶对互补10聚体引物的3'-延伸反应延迟，但在每种情况下，均获得了一些全长产物。核苷酸序列分析表明，在1,N2-ε-鸟嘌呤和HO-乙醇基鸟嘌呤对面，dGTP和dATP均发生了错配掺入，不同聚合酶之间错配程度差异很大。当评估聚合酶掺入单个dNTP的能力时，也获得了类似结果。此外，使用部分聚合酶时，在1,N2-ε-鸟嘌呤和HO-乙醇基鸟嘌呤处均检测到了-1和-2碱基移码。用大肠杆菌聚合酶I外切酶和T7聚合酶外切酶/硫氧还蛋白进行的稳态动力学实验表明，与正常的G×dCTP对相比，所有dNTP掺入的k(cat)均大幅降低，并且两种加合物的dATP和dGTP错配掺入频率都很高（与dCTP相比）。总体而言，结果表明这两种加合物对某些聚合酶具有相当大的错义编码潜力，1,N2-ε-鸟嘌呤和HO-乙醇基鸟嘌呤加合物之间存在许多差异（它们仅在是否存在水的成分上形式上有所不同），并且即使在没有外切酶活性的情况下，不同聚合酶在单个修饰碱基处dNTP的错配掺入也可能有很大差异。
