Karkas J D, Margulies L, Chargaff E
J Biol Chem. 1975 Nov 25;250(22):8657-63.
The more than 2,300-fold purification of a DNA polymerase from the embryos of Drosophila melanogaster is described. The enzyme, which forms a single band on gel electrophoresis, has a molecular weight of about 87,000 and a pH optimum of 8.5. A divalent metal is required for activity, Mg2+ being preferred with activated DNA, Mn2+ with homopolymer template-primers. The enzyme is inactivated completely by mercurials; polyamines are also inhibitory with certain templates. The most efficient template-primer is activated DNA, but homopolymers such as poly(dA)-oligo(dT), poly(A)-oligo(dT), and poly(A)-oligo(U) are also utilized with high efficiency. The purified enzyme preparations appear to be devoid of nuclease activity when assayed directly with suitable substrates. In addition, neither primer nor product is degraded after prolonged incubation with the enzyme. In accordance with previous observations on other DNA polymerases, the Drosophila enzyme can replicate single-stranded DNA only under conditions of simultaneous transcription by RNA polymerase.
本文描述了从黑腹果蝇胚胎中纯化出的一种DNA聚合酶，其纯化倍数超过2300倍。该酶在凝胶电泳上呈现单一条带，分子量约为87,000，最适pH值为8.5。酶活性需要二价金属离子，对于活化的DNA，Mg2+是首选；对于同聚物模板引物，Mn2+是首选。汞制剂可使该酶完全失活；多胺对某些模板也有抑制作用。最有效的模板引物是活化的DNA，但同聚物如聚（dA）-寡聚（dT）、聚（A）-寡聚（dT）和聚（A）-寡聚（U）也能被高效利用。当用合适的底物直接检测时，纯化的酶制剂似乎没有核酸酶活性。此外，酶与引物或产物长时间孵育后，两者均不会降解。与之前对其他DNA聚合酶的观察结果一致，果蝇酶只有在RNA聚合酶同时转录的条件下才能复制单链DNA。
