A probe for the mutagenic activity of the carcinogen 4-aminobiphenyl: synthesis and characterization of an M13mp10 genome containing the major carcinogen-DNA adduct at a unique site.
A probe for the mutagenic activity of the carcinogen 4-aminobiphenyl: synthesis and characterization of an M13mp10 genome containing the major carcinogen-DNA adduct at a unique site.
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致癌物 4-氨基联苯的诱变活性探针：在独特位点含有主要致癌物 DNA 加合物的 M13mp10 基因组的合成和表征。
DOI：
10.1021/bi00385a019
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发表时间：
1987
期刊：
影响因子：
2.9
通讯作者：
Essigmann,JM
中科院分区：
文献类型：
--
作者：
Lasko,DD;Basu,AK;Kadlubar,FF;Evans,FE;LayJr,JO;Essigmann,JM
摘要：大肠杆菌病毒M13 mpl 0的双链体基因组在一个独特的位点进行了修饰，以包含A '-（脱氧鸟苷-8-基）-4-氨基联苯（dG 8'ABP），这是人类膀胱癌致癌物4-氨基联苯的主要致癌物-DNA加合物。通过使5 ′-d（TpGpCpA）-3 ′与A-乙酰氧基-A-（三氟乙酰基）-4-氨基联苯反应，然后通过高效液相色谱法纯化主要反应产物5/-d（TpG8_ABPpCpA）-3 ′（产率15-30%），合成含有单个dG 8 ′ ABP残基的四脱氧核苷酸。通过快原子轰击质谱表征证实了结构为完整的4-氨基联苯修饰的四核苷酸，而1H核磁共振光谱确定了取代位点以及致癌物残留物和DNA碱基之间存在环堆积。5/-d（TpG 8 ′ ABPpCpA）-3 ′和5/-d（TpGpCpA）-3 ′都通过使用噬菌体T4多核苷酸激酶进行5 ′-磷酸化，并被掺入到M13 mpl 0的另外的双链体基因组中唯一位于限制性内切核酸酶Pst 1的识别位点中心的四碱基缺口中。在加合的四核苷酸的情况下，dG8_ABP位于基因组位置6270的负链中。用[~（32）P]磷酸标记四核苷酸5 ′端的实验表明：（1）在T_4DNA连接酶和ATP存在下，加合寡聚体以1000倍摩尔过量孵育时，与未加合寡聚体相比，以约30%的效率掺入到有缺口的DNA分子中。（pTpGpCpA），其以60%的连接效率掺入;（ii）将每个四核苷酸5 '端的放射性物理定位到含有Prt 1位点的限制性片段上，的基因组;（iv）在发生连接的基因组中，T4 DNA连接酶能够以至少90%的效率将修饰的和未修饰的四核苷酸共价连接到5 '和3'端的缺口结构上。还提供了证据，表明dG 8 'ABP修饰的四核苷酸对用于将其插入病毒基因组的重组DNA技术的条件是稳定的。根据这些和其他数据，dG 8 'ABP修饰的基因组被认为是用于研究大肠杆菌中的位点特异性突变的有用探针。杆菌
Revised Manuscript Received January 5, 1987 abstract: The duplex genome of Escherichia coli virus M13mpl0 was modified at a unique site to contain A'-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG8'ABP), the major carcinogen-DNA adduct of the human bladder carcinogen 4-aminobiphenyl. A tetradeoxynucleotide containing a single dG8'ABP residue was synthesized by reacting 5'-d (TpGpCpA)-3'with A-acetoxy-A-(trifluoroacetyl)-4-aminobiphenyl, followed by high-performance liquid chromatography purification of the principal reaction product 5/-d (TpG8_ABPpCpA)-3'(yield 15-30%). Characterization by fast atom bombardment mass spectrometry confirmed the structure as an intact 4-aminobiphenyl-modified tetranucleotide, while'H nuclear magnetic resonance spectroscopy established the site of substitution and the existence of ring stacking between the carcinogen residue and DNA bases. Both 5/-d (TpG8'ABPpCpA)-3/and 5/-d (TpGpCpA)-3'were 5'-phosphorylated by use of bacteriophage T4 polynucleotide kinase and were incorporated into a four-base gap uniquely positioned in the center of the recognition site for the restriction endonuclease Pstl, in an otherwise duplex genome of M13mpl0. In the case of the adducted tetranucleotide, dG8_ABP was located in the minus strand at genome position 6270. Experimentsin which the tetranucleotides were 5'end labeled with [32P] phosphate revealed the following:(i) the adducted oligomer, when incubated in a 1000-fold molar excess in the presence of T4 DNA ligase and ATP, was found to be incorporated into the gapped DNA molecules with an efficiency of approximately 30%, as compared to the unadducted d (pTpGpCpA), which was incorporated with 60% ligation efficiency;(ii) radioactivity from the 5'end of each tetranucleotide was physically mapped to a restriction fragment that containedthe Prtl site and represented 0.2% of the genome;(iii) the presence of the lesion within the Pstl recognition site inhibited the ability of Pstl to cleave the genome at this site;(iv) in genomes in which ligation occurred, T4 DNA ligase was capable of covalently joining both modified and unmodified tetranucleotides to the gapped structures on both the 5'and the 3'ends with at least 90% efficiency. Evidence also is presented showing that the dG8'ABP-modified tetranucleotide was stable to the conditions of the recombinant DNA techniques used to insert it into the viral genome. On the basis of these and other data, the dG8 ‘ABP-modified genome was judged to be a useful probe for investigation of site-specific mu-tagenesis in E. coli.
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