Fay P J, Johanson K O, McHenry C S, Bambara R A
J Biol Chem. 1982 May 25;257(10):5692-9.
Two forms of Escherichia coli DNA polymerase III, DNA polymerase III', and DNA polymerase III have been shown to synthesize DNA products via a processive mechanism with product sizes distinctive for each enzyme form. These forms of DNA polymerase III are intermediate in complexity between the core DNA polymerase III and the DNA polymerase III holoenzyme. In a previous publication (Fay, P. J., Johanson K. O., McHenry, C. S., and Bambara, R. A. (1981) J. Biol. Chem. 256, 976-983), we demonstrated that on a randomly primed fd DNA template or on an oligo(dT)10 . poly(dA) template, the DNA polymerase III holoenzyme adds more than 100 nucleotides before dissociation, whereas the core enzyme adds 10 to 15 nucleotides. Now we show that DNA polymerase III' adds 30 to 40 nucleotides before dissociation. This number can be increased to approximately 60 if spermidine is present, but it is insensitive to the presence of E. coli single-stranded DNA-binding protein. DNA polymerase III adds about 50 nucleotides before dissociation, but this value can be increased to 200 nucleotides in the presence of the binding protein. Using measurement of product sizes made on an oligo(dT)10 . poly(dA) template, reconstitution of holoenzyme activity from DNA polymerase III and the beta subunit was monitored. Finally, it is shown that the products obtained from a purified initiation complex of holoenzyme and oligo(dT)10 . poly(dA) derive solely from the holoenzyme.
已证明大肠杆菌DNA聚合酶III的两种形式，即DNA聚合酶III'和DNA聚合酶III，通过一种持续性机制合成DNA产物，每种酶形式的产物大小都不同。这些DNA聚合酶III的形式在复杂性上介于核心DNA聚合酶III和DNA聚合酶III全酶之间。在之前的一篇出版物中（费伊，P.J.，约翰森，K.O.，麦克亨利，C.S.，和班巴拉，R.A.（1981年）《生物化学杂志》256卷，976 - 983页），我们证明在随机引物的fd DNA模板或oligo(dT)10·聚(dA)模板上，DNA聚合酶III全酶在解离前添加超过100个核苷酸，而核心酶添加10到15个核苷酸。现在我们表明DNA聚合酶III'在解离前添加30到40个核苷酸。如果存在亚精胺，这个数字可以增加到大约60，但它对大肠杆菌单链DNA结合蛋白的存在不敏感。DNA聚合酶III在解离前添加约50个核苷酸，但在结合蛋白存在的情况下，这个值可以增加到200个核苷酸。使用在oligo(dT)10·聚(dA)模板上进行的产物大小测量，监测了从DNA聚合酶III和β亚基重构全酶活性的过程。最后，结果表明从全酶和oligo(dT)10·聚(dA)的纯化起始复合物获得的产物仅来自全酶。
