Preparation and characterization of a viral DNA molecule containing a site-specific 2-aminofluorene adduct: a new probe for mutagenesis by carcinogens.
Preparation and characterization of a viral DNA molecule containing a site-specific 2-aminofluorene adduct: a new probe for mutagenesis by carcinogens.
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含有位点特异性 2-氨基芴加合物的病毒 DNA 分子的制备和表征：致癌物诱变的新探针。
DOI：
10.1021/bi00350a026
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发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Romano,LJ
中科院分区：
文献类型：
--
作者：
Johnson,DL;Reid,TM;Lee,MS;King,CM;Romano,LJ
将合成的寡核苷酸七聚体S‘- atccgtc ^’与tv -乙酰-氧- 7v -（三氟乙酰基）-2-氨基芴进行体外反应，通过反相高效液相色谱（HPLC）分离得到产物。这种纯化的寡核苷酸经化学和酶分析证明是含有单个Ar-（脱氧鸟嘌呤-8-基）-2-氨基芴加合物的七聚体，然后通过将其连接到位于双链m3mp9 DNA分子负链特定位置的互补单链区域，用于定位ml3mp9基因组中假定的诱变氨基芴损伤。利用AF加合物在其限制性内切位点或附近抑制许多限制性内切酶的事实，以及AF片段应该包含在M13 mp9 DNA的印地语识别序列中，证实了该加合物在预期位置的存在。在试图用Hindi切割含有位点特异性AF加合物的Ml3 DNA时，我们发现大部分DNA仍然是圆形的，这表明AF加合物以高产量掺入到DNA分子的这个位置。该系统在体内可用于化学致癌物的诱变研究，在体外可用于研究纯化DNA代谢蛋白与含有位点特异性病变的模板的相互作用。化学致癌物的反应性亲电代谢物与DNA的亲核位点之间共价合物的形成被认为是化学致癌的关键步骤（Miller, 1978）。细胞DNA的化学修饰对细胞提出了严重的挑战，因为暴露于具有这种潜力的物质可能导致突变或细胞死亡。有大量证据表明，细胞DNA的修饰是多步致癌过程的首要事件（Weinstein, 1981; King, 1985），人们普遍认为，大多数最终致癌物是诱变剂（Ames & McCann, 1979），诱变剂通过与DNA的共价结合介导其作用（Singer & Grunberger, 1984）。
The synthetic oligonucleotide heptamer S'-ATCCGTC^'was reacted in vitro with TV-acet-oxy-7V-(trifluoroacetyl)-2-aminofluorene and the resulting product isolated by reverse-phase high-performance liquid chromatography (HPLC). This purified oligonucleotide, which was shown by chemical and enzymatic analysis to be a heptamer containing a single Ar-(deoxyguanin-8-yl)-2-aminofluorene adduct, was then used to situate the putatively mutagenic aminofluorene lesion within the genome of Ml3 mp9 by ligating it into a complementary single-stranded region located at a specific site in the negative strand of the duplex Ml 3 mp9 DNA molecule. The presence of the adduct at the anticipated location was confirmed by taking advantage of the facts that AF adducts inhibit many restriction enzymes when located in or near their restriction sites and that the AF moiety should be contained within the Hindi recognition sequence on M13 mp9 DNA. Upon attempted cleavage of the Ml3 DNA containing the site-specific AF adduct with Hindi, we find that the large majority of the DNA remained circular, demonstrating the incorporation of the AF adduct in high yield into the DNA moleculeat this location. This system should prove useful in vivo for the study of mutagenesis by chemical carcinogens and in vitro to study the interaction of purified DNA metabolizing proteins with a template containing a site-specific lesion. e formation of covalentadducts between reactive elec-trophilic metabolites of chemical carcinogens and the nu-cleophilic sites of DNA is considered to be a critical step in chemical carcinogenesis (Miller, 1978). Chemical modification of cellular DNA presents a serious challenge to cells since mutation or cell death may result upon exposure to an agent with such potential. There is substantial evidence suggesting that modification of cellular DNA is the premier event of the multistep carcinogenic process (Weinstein, 1981; King, 1985), and it is generally accepted that the majority of ultimate carcinogens are mutagens (Ames & McCann, 1979), which mediate their effect through covalent binding to DNA (Singer & Grunberger, 1984).