Wong T W, Clayton D A
J Biol Chem. 1985 Sep 25;260(21):11530-5.
A family of enzymatic activities isolated from human mitochondria is capable of initiating DNA replication on single-stranded templates. The principal enzymes include at least a primase and DNA polymerase gamma and require that rNTPs as well as dNTPs be present in the reaction mixture. Poly(dC) and poly(dT), as well as M13 phage DNA, are excellent templates for the primase activity. A single-stranded DNA containing the cloned origin of mitochondrial light-strand synthesis can be a more efficient template than M13 phage DNA alone. Primase and DNA polymerase activities were separated from each other by sedimentation in a glycerol density gradient. Using M13 phage DNA as template, these mitochondrial enzymes synthesize RNA primers that are 9 to 12 nucleotides in size and are covalently linked to nascent DNA. The formation of primers appears to be the rate-limiting step in the replication process. Replication of M13 DNA is sensitive to N-ethylmaleimide and dideoxynucleoside triphosphates, but insensitive to rifampicin, alpha-amanitin, and aphidicolin.
从人线粒体中分离出的一系列酶活性能够在单链模板上启动DNA复制。主要酶类至少包括一种引发酶和DNA聚合酶γ，并且要求反应混合物中同时存在rNTP和dNTP。聚（dC）和聚（dT）以及M13噬菌体DNA是引发酶活性的优良模板。包含克隆的线粒体轻链合成起始位点的单链DNA可能比单独的M13噬菌体DNA更有效的模板。通过在甘油密度梯度中沉降，可将引发酶和DNA聚合酶活性彼此分离。以M13噬菌体DNA为模板，这些线粒体酶合成大小为9至12个核苷酸且与新生DNA共价连接的RNA引物。引物的形成似乎是复制过程中的限速步骤。M13 DNA的复制对N-乙基马来酰亚胺和双脱氧核苷三磷酸敏感，但对利福平、α-鹅膏蕈碱和阿非迪霉素不敏感。
