Watabe K, Leusch M, Ito J
Proc Natl Acad Sci U S A. 1984 Sep;81(17):5374-8. doi: 10.1073/pnas.81.17.5374.
phi 29 DNA replication is initiated by the formation of a covalent complex between the viral-coded terminal protein and dAMP (TP-dAMP). This initiation reaction system has been reconstituted from two phage-encoded proteins, the terminal protein and DNA polymerase. The phi 29 DNA polymerase was purified from phage-infected cells by using poly(dA) X p(dT)12-18 as an assay template. The purified polymerase has an apparent molecular mass of 68 kDa in its native form and it appears to function as a monomer. The terminal protein was purified to homogeneity from Escherichia coli cells harboring a cloned plasmid that contained a phi 29 gene 3 segment. The molecular mass of the purified terminal protein was about 30 kDa in both the denatured and the native form. The protein apparently functions as a monomer. When the terminal protein and DNA polymerase were incubated in the presence of dATP, Mg2+, and phi 29 DNA-protein as template, the terminal protein bound covalently to dAMP. This reaction did not require ATP. In addition, these two purified fractions catalyzed DNA chain elongation from both ends of phi 29 DNA, yielding the expected 9- to 12-base fragment when assayed in the presence of 2',3'-dideoxycytidine triphosphate. These results indicate that phi 29 DNA polymerase catalyzes formation of the terminal protein-dAMP complex and can also catalyze chain elongation at least 9-12 bases from both ends of phi 29 DNA.
phi 29 DNA复制由病毒编码的末端蛋白与dAMP（TP-dAMP）形成共价复合物引发。该起始反应系统已由两种噬菌体编码蛋白，即末端蛋白和DNA聚合酶重构而成。phi 29 DNA聚合酶通过使用聚（dA）X p（dT）12 - 18作为检测模板从噬菌体感染的细胞中纯化得到。纯化后的聚合酶天然形式下的表观分子量为68 kDa，且似乎以单体形式发挥作用。末端蛋白从携带含有phi 29基因3片段的克隆质粒的大肠杆菌细胞中纯化至同质。纯化后的末端蛋白在变性和天然形式下的分子量均约为30 kDa。该蛋白显然以单体形式发挥作用。当末端蛋白和DNA聚合酶在dATP、Mg2 +以及phi 29 DNA - 蛋白作为模板存在的情况下孵育时，末端蛋白与dAMP共价结合。该反应不需要ATP。此外，这两个纯化组分催化了phi 29 DNA两端的DNA链延伸，在2'，3'-双脱氧胞苷三磷酸存在下进行检测时产生了预期的9至12个碱基的片段。这些结果表明，phi 29 DNA聚合酶催化末端蛋白 - dAMP复合物的形成，并且还能催化phi 29 DNA两端至少9至12个碱基的链延伸。
