基于脱氧核苷三磷酸反应性类似物的催化活性对DNA聚合酶α-引物酶复合物进行光亲和标记。

Photoaffinity labeling of DNA polymerase alpha DNA primase complex based on the catalytic competence of a dNTP reactive analog.

作者信息

Doronin S V, Dobrikov M I, Lavrik O I

机构信息

Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of the Russian Academy of Sciences.

出版信息

FEBS Lett. 1992 Nov 16;313(1):31-3. doi: 10.1016/0014-5793(92)81177-n.

DOI:10.1016/0014-5793(92)81177-n

PMID:1426265

Abstract

FABdCTP was found to be a substrate of DNA polymerization catalyzed by a DNA polymerase alpha-DNA primase complex on the 5'-GTGAGTAAGTGGAGTTTGGCACGAT-3' template and 3'-CTCAAACCGT-5' primer. After complete primer extension in the presence of FABdCTP under UV-irradiation of the reaction mixture, 70% of the template was covalently linked to the primer. Labeling of the 165 kDa subunit of the DNA polymerase alpha, 59 kDa and 49 kDa subunits of the DNA primase and an unknown protein with apparent molecular weight of 31 kDa was observed. By another way of protein labeling FABdCTP was covalently bound to the subunits of the enzyme under UV irradiation and then this moiety was introduced into the 3'-end of the 5'-[32P]primer by the catalytic activity of DNA polymerase or DNA primase. In this case covalent labeling of the 165 kDa, 49 kDa and 31 kDa subunits was observed.

摘要

研究发现，FABdCTP是DNA聚合酶α-DNA引发酶复合物在5'-GTGAGTAAGTGGAGTTTGGCACGAT-3'模板和3'-CTCAAACCGT-5'引物上催化DNA聚合反应的底物。在反应混合物紫外线照射下，存在FABdCTP时引物完全延伸后，70%的模板与引物共价连接。观察到DNA聚合酶α的165 kDa亚基、DNA引发酶的59 kDa和49 kDa亚基以及表观分子量为31 kDa的未知蛋白被标记。通过另一种蛋白质标记方法，FABdCTP在紫外线照射下与酶的亚基共价结合，然后该部分通过DNA聚合酶或DNA引发酶的催化活性被引入到5'-[32P]引物的3'-末端。在这种情况下，观察到165 kDa、49 kDa和31 kDa亚基的共价标记。