Brooks M, Dumas L B
Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60201.
J Biol Chem. 1989 Feb 25;264(6):3602-10.
We have utilized immunoaffinity chromatography as a means of efficiently isolating a stable yeast DNA primase from the DNA primase-DNA polymerase complex, allowing identification of the polypeptides associated with this DNA primase activity and comparison of its enzymatic properties with those of the larger protein complex. A mouse monoclonal antibody specifically recognizing the DNA polymerase subunit was used to purify the complex. Stable DNA primase was subsequently separated from the complex in high yield. The highly purified protein fraction which bound to the DNA polymerase antibody column consisted of polypeptides with apparent molecular masses of 180, 86, 70, 58, 49, and 47 kDa. DNA primase activity eluted with a fraction containing only the 58-, 49-, and 47-kDa polypeptides. Partial chemical cleavage analysis of these three proteins demonstrated that the 49- and 47-kDa polypeptides are structurally related while the 58-kDa protein is unrelated to the other two. A DNA primase inhibitory monoclonal antibody was able to inhibit the activity of the purified DNA primase as well as the activity of the enzyme in the larger complex. In immunoprecipitation experiments, all three polypeptides were found in the immune complex. Thus, these three polypeptides are sufficient for DNA primase activity. In reactions using ribonucleotide substrates and natural as well as synthetic DNA templates, the purified DNA primase exhibited the same precise synthesis of unit length oligomers as did the larger protein complex and was able to extend these RNA oligomers by one additional unit length. An examination of the effects of deoxynucleotides on these DNA primase-catalyzed reactions revealed that the yeast DNA primase is an RNA-polymerizing enzyme and lacks significant DNA-polymerizing activity under the conditions tested.
我们利用免疫亲和层析法，从DNA引物酶 - DNA聚合酶复合物中高效分离出一种稳定的酵母DNA引物酶，从而能够鉴定与该DNA引物酶活性相关的多肽，并将其酶学性质与较大的蛋白质复合物的酶学性质进行比较。使用一种特异性识别DNA聚合酶亚基的小鼠单克隆抗体来纯化该复合物。随后以高产率从复合物中分离出稳定的DNA引物酶。与DNA聚合酶抗体柱结合的高度纯化的蛋白质组分由表观分子量分别为180、86、70、58、49和47 kDa的多肽组成。DNA引物酶活性随仅含有58、49和47 kDa多肽的组分洗脱。对这三种蛋白质的部分化学裂解分析表明，49 kDa和47 kDa的多肽在结构上相关，而58 kDa的蛋白质与其他两种无关。一种DNA引物酶抑制性单克隆抗体能够抑制纯化的DNA引物酶的活性以及较大复合物中该酶的活性。在免疫沉淀实验中，在免疫复合物中发现了所有三种多肽。因此，这三种多肽足以产生DNA引物酶活性。在使用核糖核苷酸底物以及天然和合成DNA模板的反应中，纯化的DNA引物酶与较大的蛋白质复合物一样，表现出相同精确合成单位长度寡聚物的能力，并且能够将这些RNA寡聚物再延长一个单位长度。对脱氧核苷酸对这些DNA引物酶催化反应的影响进行的研究表明，酵母DNA引物酶是一种RNA聚合酶，在测试条件下缺乏显著的DNA聚合活性。
