A new sensitive 32P-postlabeling assay based on the specific enzymatic conversion of bulky DNA lesions to radiolabeled dinucleotides and nucleoside 5'-monophosphates.
A new sensitive 32P-postlabeling assay based on the specific enzymatic conversion of bulky DNA lesions to radiolabeled dinucleotides and nucleoside 5'-monophosphates.
复制标题
一种新的灵敏 32P 标记后测定，基于大体积 DNA 损伤到放射性标记二核苷酸和核苷 5-单磷酸的特异性酶促转化。
DOI：
10.1093/carcin/10.7.1231
复制
发表时间：
1989
期刊：
影响因子：
4.7
通讯作者：
Putman,KL
中科院分区：
文献类型：
--
作者：
Randerath,K;Randerath,E;Danna,TF;vanGolen,L;Putman,KL
一种新的DNA加合物的敏感32p标记后检测方法已经开发出来，其中DNA最初由核酸酶P1和前列腺酸性磷酸酶水解，而不是像以前的标记后程序那样由微球菌核酸酶和脾脏磷酸二酯酶水解。当DNA含有庞大的加合物，X1, X2,.....Xn，在pH为5时被核酸酶P1消化，正常核苷酸被释放为5'-单磷酸，pN，而加合物被切除为5‘-磷酸化二核苷酸，pX1pN，因为X 3’侧的核苷酸间键抵抗核酸酶P1的攻击。将前列腺酸性磷酸酶添加到这样的消化酶中，导致核苷酸的5‘-去磷酸化成为正常核苷，N和内合的二核苷酸，X1pN，携带一个5’端游离羟基。通过T4多核苷酸激酶催化的[32P]磷酸转移，[γ-32P]ATP将二核苷酸（非核苷）转化为5'-32P标记的二核苷酸[32P]pX1pN。在聚乙烯亚胺-纤维素阴离子交换薄层色谱上，标记的二核苷酸加合物产生特征的自放射指纹图谱。或者，它们被蛇毒磷酸二酯酶进一步消化，产生5'-单磷酸，（32P） px1和pN。单磷酸加合物的TLC谱与二核苷酸的TLC谱不同。这些反应为新的32P后标记方案提供了基础，本文将其与先前报道的以5'-32P标记的3',5'-二磷酸盐，[32P]pX1p形式生成加合物的方案进行了比较。结果表明，同一加合物的三种不同类型的32p后标记衍生物的可用性有助于不同外源和内源DNA加合物的分析和色谱表征。
A new sensitive32P-postlabeling assay for DNA adducts has been developed in which DNA is hydrolyzed initially by nuclease P1 and prostatic acid phosphatase instead of micrococcal nuclease and spleen phosphodiesterase as employed in previous postlabeling procedures. When DNA containing bulky adducts, X1, X2,.....Xn, is digested with nuclease P1 at pH 5, normal nucleotides are released as 5'-monophosphates, pN, while adducts are excised as 5'-phosphorylated dinucleotides, pX1pN, because internucleotide linkages on the 3' side of X resist attack by nuclease P1. Addition of prostatic acid phosphatase to such a digest results in 5'-dephosphorylation of the nucleotides to normal nucleosides, N, and adducted dinucleotides, X1pN, carrying a 5'-terminal free hydroxyl group. The dinucleotides but not nucleosides are converted to 5'-32P-labeled dinucleotides, [32P]pX1pN, by T4 polynucleotide kinase-catalyzed [32P]phosphate transfer from [γ-32P]ATP. Upon mapping on polyethyleneimine-cellulose anion-exchange TLC, the labeled dinucleotide adducts produce characteristic autoradiographic fingerprints. Alternatively, they are further digested with snake venom phosphodiesterase to yield 5'-monophosphates, (32P)pX1and pN. TLC profiles of the monophosphate adducts are distinct from those of the dinucleotides. These reactions provide the basis of the new32P-postlabeling scheme, which is compared in this paper with a previously reported protocol yielding adducts in the form of 5'-32P-labeled 3', 5'-bisphosphates, [32P]pX1p. The results show that the availability of three different types of32P-postlabeled derivatives for the same adduct aids in the analysis and chromatographic characterization of DNA adducts from diverse exogenous and endogenous sources.