Wilson F E, Sugino A
J Biol Chem. 1985 Jul 5;260(13):8173-81.
A primase activity which permits DNA synthesis by yeast DNA polymerase I on a single-stranded circular phi X174 or M13 DNA or on poly(dT)n has been extensively purified by fractionation of a yeast enzyme extract which supports in vitro replication of the yeast 2-microns plasmid DNA (Kojo, H., Greenberg, B. D., and Sugino, A. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 7261-7265). Most of this DNA primase activity was separated from DNA polymerase activity, although a small amount remained associated with DNA polymerase I. The primase, active as a monomer, has a molecular weight of about 60,000. The primase synthesizes oligoribonucleotides of discrete size, mainly eight or nine nucleotides, in the presence of single-stranded template DNA and ribonucleoside 5'-triphosphates; it utilizes deoxyribonucleoside 5'-triphosphates as substrate with 10-fold lower efficiency. Product size, chromatographic properties, alpha-amanitin resistance, and molecular weight of the primase activity distinguish it from RNA polymerases I, II, and III. The DNA products synthesized by both primase and DNA polymerase I on a single-stranded DNA template were 200-500 nucleotides long and covalently linked to oligoribonucleotides at their 5'-ends. Addition of yeast single-stranded DNA-binding protein (Arendes, J., Kim, K. C., and Sugino, A. (1983) Proc. Natl. Acad. Sci. U.S. A. 80, 673-677) stimulated the DNA synthesis 2-3-fold.
一种能使酵母DNA聚合酶I在单链环状φX174或M13 DNA或聚（dT）n上进行DNA合成的引发酶活性，已通过对支持酵母2μm质粒DNA体外复制的酵母酶提取物进行分级分离而得到广泛纯化（小条浩二、格林伯格、B.D.和杉野明（1981年），《美国国家科学院院刊》78卷，7261 - 7265页）。这种DNA引发酶活性的大部分与DNA聚合酶活性分离，尽管仍有少量与DNA聚合酶I相关。该引发酶以单体形式具有活性，分子量约为60,000。在单链模板DNA和核糖核苷5'-三磷酸存在的情况下，引发酶合成离散大小的寡核糖核苷酸，主要是八个或九个核苷酸；它以低10倍的效率利用脱氧核糖核苷5'-三磷酸作为底物。引发酶活性的产物大小、色谱性质、α-鹅膏蕈碱抗性和分子量使其与RNA聚合酶I、II和III区分开来。引发酶和DNA聚合酶I在单链DNA模板上合成的DNA产物长度为200 - 500个核苷酸，并且在其5'-末端与寡核糖核苷酸共价连接。添加酵母单链DNA结合蛋白（阿伦德斯、J.、金、K.C.和杉野明（1983年），《美国国家科学院院刊》80卷，673 - 677页）可使DNA合成提高2 - 3倍。
