Sequence selectivity in the reaction of optically active hydrocarbon dihydrodiol epoxides with rat H-ras DNA.
Sequence selectivity in the reaction of optically active hydrocarbon dihydrodiol epoxides with rat H-ras DNA.
复制标题
光学活性烃二氢二醇环氧化物与大鼠 H-ras DNA 反应中的序列选择性。
DOI：
10.1021/tx00007a002
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发表时间：
1989
影响因子：
4.1
通讯作者：
Dipple,A
中科院分区：
文献类型：
--
作者：
Reardon,DB;Bigger,CA;Strandberg,J;Yagi,H;Jerina,DM;Dipple,A
图 2. 放射自显影照片显示了由图 1 中描述的光学活性二氢二醇环氧化物对 ras DNA 序列进行化学修饰而引起的 DNA 聚合酶（测序酶）停滞位点。图 AD 显示了有义链和反义链中与密码子 61 相邻的序列，而图 E 和 F 显示了有义链中与密码子 12 相邻的序列。用于处理DNA的烃衍生物在图中标出了密码子61和密码子12的位置。符号C、G、T和A表示未修饰的pAL-7模板链的序列。 pAL-7 的等分试样 [此 pBR322 衍生物包含 2.9 kb f/mdIII DNA 片段，包含正常大鼠 H-ras 基因除前 12 个核苷酸 (16) 之外的所有编码序列，是 NCI-Frederick 癌症研究机构的 Mariano Barbacid 博士赠送的礼物]（0.8 或 0.2 mg/mL，溶于 0.01 M Tris-0.001 M EDTA 缓冲液（pH 7.4，分别用于溶剂中的 BcPh 和 BaP 衍生物）分别用 BcPhDE (25)（图 1）以 7.5、7.2、11.7 和 6.6 ng/* ig 的 DNA 处理 (+)-1、(-)-1、(+)-2 和 (—)-2 异构体，以便每 200 个核苷酸中大约有 1 个被修改后，每种情况下乙腈的最终浓度均为 22%。通过使用68和208ng/ng的(+)-2和(-)-2对映异构体的DNA获得了BaP衍生物的类似水平的DNA修饰(NCI化学致癌物存储库，NCI，Bethesda，MD)。 BaP衍生物溶于四氢呋喃/三乙胺(19:1)中，缓冲液中有机溶剂的最终浓度为22%。反应在37°C、黑暗中进行3小时。作为对照，一些 DNA 单独用溶剂处理。用0.1体积的2M NaOH-0.002M EDTA使处理的pAL-7变性，5分钟后，添加等摩尔量的N-标记引物，然后用0.1体积的2M乙酸铵(pH 4.5)中和。用乙醇沉淀并干燥后，将 DNA 沉淀重悬于 0.03 M TrisHCl（pH 7.5）、0.015 M MgCl2、0.037 M NaCl 和 0.0075 M 二硫苏糖醇（7 nL/ng DNA）中。向7nh的DNA溶液中添加dATP、dGTP、dCTP和TTP各80/jM的核苷酸溶液(5μL)和6.25单位的测序酶(United States Biochemical Corp.，Cleveland，OH)(4mL)。在37℃下5分钟后，添加4×iL终止溶液(95％甲酰胺、20mM EDTA、0.05％溴酚蓝和0.05％二甲苯氰FF)。将具有等量放射性的等分试样加热至90°C 3分钟，并在6%变性聚丙烯酰胺凝胶中进行电泳。未修饰 DNA 的测序遵循 Zagursky 等人 (26) 的一般方法，基本上如上所述，只是终止反应遵循测序方案。密码子 61 是 TTG，没有证据表明 BaP 衍生物（图 2B）或 BcPh 衍生物（图 2D）有任何实质性反应，尽管在两种情况下都可以在鸟嘌呤附近看到弱捕获位点。还探讨了密码子 12 GGA 序列区域中反应的序列特异性（图 2E、F），但由于补体包含所有嘧啶，这些嘧啶对这些衍生物不具有反应性，因此未检查反义链。所有六种二氢二醇环氧化物均在密码子 12 的 GGA 处发生反应。用 BcPh 衍生物形成的条带强度与其他地方的许多其他条带的强度相似，表明选择性很小。该密码子处的 BaP 诱导条带不是凝胶上最强烈的条带，但它们比许多其他鸟嘌呤处的条带更强烈，表明该密码子的反应具有一定程度的选择性。我们的研究结果表明 BaP 代谢物表现出……
Figure 2. Autoradiographs showingsites of arrest of DNA polymerase (Sequenase) caused by chemical modification of ras DNA sequences by the optically active dihydrodiol epoxides described in Figure 1. Panels AD show sequences adjacent to codon 61 in the sense and antisense strands, while panels E and F show the sequence adjacent to codon 12 in the sense strand. The hydrocarbon derivatives used to treat the DNA are indicated on the figure as are the locations of codon 61 and codon 12. The symbols C, G, T, and A indicate the sequence of the template strand of unmodified pAL-7. Aliquots of pAL-7 [this pBR322 derivative, which contains a 2.9-kb f/mdIII DNA fragmentencompassing all the coding sequences of the normal rat H-ras gene except the first 12 nucleotides (16), was a gift from Dr. Mariano Barbacid, NCI-Frederick Cancer Research Facility](0.8 or 0.2 mg/mL in 0.01 M Tris-0.001 M EDTA buffer, pH 7.4, for BcPh and BaP derivatives in solvent, respectively) were treated with BcPhDEs (25)(Figure 1) at 7.5, 7.2, 11.7, and 6.6 ng/* ig of DNA for the (+)-l,(-)-l,(+)-2, and (—)-2 isomers, respectively, so that approximately 1 in every 200 nucleotides was modified and the final concentration of acetonitrile was 22% in each case. Similar levels of DNA modification by theBaP derivatives were obtained by using 68 and 208 ng/ng of DNA of the (+)-2 and (-)-2 enantiomers (NCI ChemicalCarcinogen Repository, NCI, Bethesda, MD). The BaP derivatives were in tetrahydrofuran/triethylamine (19: 1), and the final concentration of organic solvent in buffer was 22%. Reactions were for 3 h at 37 C in the dark. As a control, some DNA was treated with the solvents alone. TreatedpAL-7 was denatured with 0.1 volume of 2 M NaOH-0.002 M EDTA, and after 5 min, an equimolar amount of^-labeled primer was added prior to neutralization with 0.1 volume of 2 M ammonium acetate, pH 4.5. After precipitation with ethanol and drying, the DNA pellet was resuspended in 0.03 M TrisHCl, pH 7.5, 0.015 M MgCl2, 0.037 M NaCl, and 0.0075 M dithiothreitol (7 nL/ng of DNA). To 7 nh of DNA solution was added nucleotide solution, 80/jM in each of dATP, dGTP, dCTP, and TTP (5 fiL), and 6.25 units of Sequenase (United States Biochemical Corp., Cleveland, OH)(4 mL). After 5 min at37 C, 4* iL of stop solution (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol FF) was added. Aliquots with equal amounts of radioactivity were heated to90 C for 3 min and electrophoresed in 6% denaturing polyacrylamide gels. Sequencing of unmodifiedDNA followed the general approach of Zagursky et al.(26) and was essentially as described above except that the termination reactions followed the Sequenase protocol. codon 61 is TTG, no evidence for any substantial reaction was seen with the BaP derivatives (Figure 2B) or the BcPh derivatives (Figure 2D), though weak arrest sites could be seen immediately adjacent to the guanine in both cases. The sequence specificity of reactions in the region of the codon 12 GGA sequence was also explored (Figure 2E, F), but since thecomplement contains all pyrimidines, which are not reactive toward these derivatives, the antisense strand was not examined. All six dihydrodiol epoxides reacted at the GGA of codon 12. The intensities of the bands formed with the BcPh derivatives were similar to those of many other bands elsewhere, indicating little se-lectivity. The BaP-inducedbands at this codon were not the most intense bands on the gel, but they were more intense than bands at many other guanines, suggesting some degree of selectivity in reaction at this codon. Our findings indicate that the BaP metabolites exhibit …