Mechanism of frameshift (deletion) generated by acetylaminofluorene-derived DNA adducts in vitro.
Mechanism of frameshift (deletion) generated by acetylaminofluorene-derived DNA adducts in vitro.
复制标题
乙酰氨基芴衍生的 DNA 加合物在体外产生移码（缺失）的机制。
DOI：
10.1021/bi048087e
复制
发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Grollman,ArthurP
中科院分区：
文献类型：
--
作者：
Shibutani,Shinya;Suzuki,Naomi;Grollman,ArthurP
我们研究了乙酰氨基芴（AAF-）衍生的DNA加合物诱导移码（缺失）突变的机制。将dG-AAF修饰的寡脱氧核苷酸与32 P标记的13-mer引物退火，然后在大肠杆菌DNA聚合酶I的无3 '→ 5'外切核酸酶的Klenow片段催化下进行引物延伸反应。当位于dG-AAF对面的dNMP可以与其5'侧翼位置的互补碱基配对时，以高频率产生单碱基缺失。类似地，当互补碱基位于dG-AAF的5'的两个位置时，发生两个碱基缺失。在dG-AAF对面碱基插入的相对频率为dCMP > dAMP > dGMP > dTMP，dNTP对面碱基插入的相对频率与移码缺失的形成无关。当设计用于诱导三个碱基缺失的模板用于由exo-Klenow片段催化的跨损伤合成时，形成了预期的三个碱基缺失。当含有位于损伤5'端的重复碱基的dG-AAF修饰的模板与具有位于损伤对面的互补dNMP的引物退火时，插入dG-AAF对面的dNMP倾向于与损伤5'端的互补碱基配对，从而形成较短的缺失。总之，这些结果支持Shibutani和Grollman早先提出的移码缺失的分子机制，其中直接碱基插入先于错配[（1993）J.Biol.Chem.268，11703]。
We have investigated the mechanism of frameshift (deletion) mutagenesis induced by acetylaminofluorene- (AAF-) derived DNA adducts. dG-AAF-modified oligodeoxynucleotides, with different bases positioned 5‘ to the lesion, were annealed to32P-labeled 13-mer primers and then used in primer extension reactions catalyzed by the 3‘→5‘ exonuclease-free Klenow fragment ofEscherichia coliDNA polymerase I. When the dNMP positioned opposite dG-AAF could pair with its complementary base at the 5‘ flanking position, single-base deletions were produced at high frequency. Similarly, when the complementary base was two positions 5‘ to the dG-AAF, two-base deletions occurred. The relative frequency of base insertions opposite dG-AAF followed the order dCMP > dAMP > dGMP > dTMP; the frequency of dNTP insertion opposite the lesion paralleled the formation of frameshift deletions. When a template designed to induce three-base deletions was used for translesion synthesis catalyzed by the exo-Klenow fragment, the expected three-base deletion was formed. When dG-AAF-modified templates containing iterated bases 5‘ to the lesion were annealed to primers with the complementary dNMP positioned opposite the lesion, the dNMP inserted opposite the dG-AAF tended to pair with the complementary base 5‘ to the lesion, thereby forming shorter deletions. Taken together, these results support the molecular mechanism for frameshift deletion proposed earlier by Shibutani and Grollman in which direct base insertion precedes misalignment [(1993)J. Biol. Chem.268, 11703].