DNA polymerase-mediated nucleotide incorporation adjacent to hydrocarbon-deoxyadenosine and hydrocarbon-deoxyguanosine adducts.
DNA polymerase-mediated nucleotide incorporation adjacent to hydrocarbon-deoxyadenosine and hydrocarbon-deoxyguanosine adducts.
复制标题
DNA 聚合酶介导的核苷酸掺入邻近烃-脱氧腺苷和烃-脱氧鸟苷加合物。
DOI：
10.1093/carcin/12.9.1659
复制
发表时间：
1991
期刊：
影响因子：
4.7
通讯作者：
Dipple,A
中科院分区：
文献类型：
--
作者：
Hruszkewycz,AM;Canella,KA;Dipple,A
为了研究DNA加合物对DNA聚合酶在3′端相邻碱基处掺入核苷酸的影响，制备了合成的寡核苷酸（16聚体），其在3′端的13位含有嘌呤，在12位含有4个可能的碱基中的任何一个，并用7-溴甲基苯并[a]蒽读取。使用HPLC，未修饰的寡核苷酸与在腺嘌呤或鸟嘌呤残基处含有单一加合物的寡核苷酸分离。这些产物用32 P 5′-末端标记引物（11 mer）退火，并在脱氧核糖核苷5′-三磷酸存在下与修饰的T7 DNA聚合酶（Sequenase，2.0版）孵育。凝胶电泳分析表明，未修饰的寡核苷酸模板允许引物快速延伸至模板的全长。然而，加合物的存在导致引物延伸在加合物的碱基3′处停止。虽然正确的碱基配对发生在这个终止位点与大多数加合物模板，有一个高频率的错误掺入鸟嘌呤相对于胸腺嘧啶位于3′腺嘌呤加合物。这一结果表明，一些庞大的致癌物-DNA加合物可能会导致在相邻碱基的碱基错配。
To examine the effect of DNA adducts on nucleotide incorporation by DNA polymerase at 3′ neighboring bases, synthetic oligonucleotides (16mers) containing a purine at position 13 from the 3′ end and any one of the four possible bases at psition 12 were prepared and readed with 7-bromomethylbenz[a]anthracene. Using HPLC, unmodified oligonucleotide was separated fhm oligonucleotide containtng a single adduct, at either an adenine or a guanine residue. These products were annealed with a32P 5′-end labeled primer (llmer) and incubated with modified T7 DNA polymerase (Sequenase, version 2.0) in the presence of deoxyribonucleoside 5′-triphosphates. Analysis by gel electrophoresis showed that unmodified oligonucleotide template allowed the primer to be rapidly extended to the entire length of the template. However, the presence of an adduct caused primer extension to stop at the base 3′ to the adduct. While correct base pairing occurred at this termination site with most adducted templates, there was a high frequency of misincorporation of guanine opposite a thymine located 3′ to an adenine adducts. This result suggests that some bulky carcinogen-DNA adducts may lead to base mismatches at neighboring bases.