Philippe M, Rossignol J M, De Recondo A M
Biochemistry. 1986 Apr 8;25(7):1611-5. doi: 10.1021/bi00355a024.
A primase activity associated to DNA polymerase alpha from rat liver is described. Both activities were absent in normal adult rat liver but were concomitantly induced after partial hepatectomy. As previously shown for polymerase alpha and DNA topoisomerase II, primase activity reached a maximum value 40-43 h after the partial removal of the liver. Primase activity was shown to catalyze dNMP incorporation on unprimed single-stranded DNA template (M13 DNA) in the presence of rNTP. The activity was not detectable on poly(dA) or poly(dG) but was efficient on poly(dT) or poly(dC). However, the reliability of the primase assay in the presence of poly(dC) was dependent upon the degree of purification of the enzyme. The ribo primers were about 10 nucleotides long, and the reaction was completely independent of alpha-amanitin, a strong inhibitor of RNA polymerases II and III. Primase and polymerase were found tightly associated. A cosedimentation on a 5-20% sucrose gradient was always obtained, independent of the ionic strength. There was also a close coincidence between alpha-polymerase and primase activities during phosphocellulose, hydroxylapatite, and single-stranded DNA Ultrogel chromatography. It has been previously demonstrated by us and others that primase and alpha-polymerase are on separated polypeptides. The association of two activities in the replication complex and the conditions allowing their separation are discussed.
本文描述了与大鼠肝脏DNA聚合酶α相关的引发酶活性。在正常成年大鼠肝脏中这两种活性均不存在，但在部分肝切除术后它们会同时被诱导产生。正如之前对聚合酶α和DNA拓扑异构酶II的研究表明，引发酶活性在部分肝脏切除后40 - 43小时达到最大值。已证明引发酶活性在核糖核苷三磷酸（rNTP）存在的情况下，能催化脱氧单磷酸核苷酸（dNMP）掺入未引发的单链DNA模板（M13 DNA）中。在聚（dA）或聚（dG）上检测不到该活性，但在聚（dT）或聚（dC）上该活性有效。然而，在聚（dC）存在下引发酶测定的可靠性取决于酶的纯化程度。核糖引物长度约为10个核苷酸，并且该反应完全不受α-鹅膏蕈碱（一种RNA聚合酶II和III的强抑制剂）的影响。发现引发酶与聚合酶紧密相关。在5 - 20%的蔗糖梯度上总是能得到共沉降，这与离子强度无关。在磷酸纤维素、羟基磷灰石和单链DNA Ultrogel柱层析过程中，α-聚合酶和引发酶活性也有密切的一致性。我们和其他人之前已证明引发酶和α-聚合酶存在于不同的多肽上。本文讨论了复制复合物中两种活性的关联以及使其分离的条件。
