Engler M J, Lechner R L, Richardson C C
J Biol Chem. 1983 Sep 25;258(18):11165-73.
The DNA polymerase induced by bacteriophage T7 can be isolated in two different forms. The distinguishing properties are: 1) the specific activities of the associated 3' to 5' single- and double-stranded DNA exonuclease activities, 2) the ability to catalyze DNA synthesis and strand displacement at nicks, and 3) the degree of stimulation of DNA synthesis on nicked, duplex DNAs by the gene 4 protein of phage T7. Form I is obtained when purification is carried out in the absence of EDTA while Form II is obtained if all purification steps are carried out in the presence of 0.1 mM EDTA. Form I has low levels of both exonuclease activities, less than 5% of those of Form II. Form I can initiate DNA synthesis at nicks leading to strand displacement, a consequence of which is its ability to be stimulated manyfold by the helicase activity of gene 4 protein on nicked, duplex templates. On the other hand, Form II cannot initiate synthesis at nicks even in the presence of gene 4 protein. In keeping with its higher exonuclease activities, Form II of T7 DNA polymerase has higher turnover of nucleotides activity (5-fold higher than Form I) and exhibits greater fidelity of nucleotide incorporation, as indicated by the rate of incorporation of 2-aminopurine deoxynucleoside monophosphate. Both forms of T7 DNA polymerase exhibit higher fidelity of nucleotide incorporation than bacteriophage T4 DNA polymerase. In the absence of EDTA or in the presence of FeSO4 or CaCl2, Form II irreversibly converts to Form I. The physical difference between the two forms is not known. No difference in molecular weight can be detected between the corresponding subunits of each form of T7 DNA polymerase as measured by gel electrophoresis in the presence of sodium dodecyl sulfate.
由噬菌体T7诱导产生的DNA聚合酶可以以两种不同形式分离得到。其区别特性如下：1）相关的3'至5'单链和双链DNA外切核酸酶活性的比活性；2）催化切口处DNA合成和链置换的能力；3）噬菌体T7基因4蛋白对带切口双链DNA上DNA合成的刺激程度。在没有EDTA的情况下进行纯化可得到I型，而如果所有纯化步骤都在0.1 mM EDTA存在下进行，则可得到II型。I型的两种外切核酸酶活性水平都很低，不到II型的5%。I型可以在切口处起始DNA合成并导致链置换，其结果是它能被基因4蛋白在带切口双链模板上的解旋酶活性刺激许多倍。另一方面，即使存在基因4蛋白，II型也不能在切口处起始合成。与较高的外切核酸酶活性一致，T7 DNA聚合酶的II型具有更高的核苷酸周转活性（比I型高5倍），并且如2-氨基嘌呤脱氧核苷单磷酸的掺入速率所示，表现出更高的核苷酸掺入保真度。T7 DNA聚合酶的两种形式都比噬菌体T4 DNA聚合酶表现出更高的核苷酸掺入保真度。在没有EDTA的情况下，或在存在硫酸亚铁或氯化钙的情况下，II型会不可逆地转化为I型。两种形式之间的物理差异尚不清楚。通过在十二烷基硫酸钠存在下进行凝胶电泳测量，每种形式的T7 DNA聚合酶相应亚基之间未检测到分子量差异。
