Cha T A, Alberts B M
Department of Biochemistry and Biophysics, University of California, San Francisco 94143.
J Biol Chem. 1989 Jul 25;264(21):12220-5.
Seven bacteriophage T4-encoded proteins reconstitute a DNA replication apparatus that catalyzes coupled leading and lagging strand DNA synthesis at a replication fork in vitro. The proteins involved are the T4 DNA polymerase holoenzyme (the products of T4 genes 43, 44/62, and 45), a helix-destabilizing (SSB) protein (gene 32 protein), and the T4 primosome which is composed of a DNA helicase (gene 41 protein) and a primase (gene 61 protein). We show here that the presence of 41 protein on the lagging strand of the fork enables the polymerase holoenzyme to catalyze leading strand DNA synthesis at a maximum rate and with high processivity. This leading strand synthesis is unaffected by the addition of either the gene 32 or the gene 61 protein; the 41 protein cannot be replaced by the dda protein, a second T4-encoded DNA helicase. When the 61 protein is added to the 41 protein to complete the primosome, Okazaki fragment synthesis on the lagging strand accompanies leading strand DNA synthesis in this system even in the absence of the 32 protein. However, the addition of 32 protein decreases the size of the Okazaki fragments made, as expected for an increase in the lagging strand polymerization rate at a fork that has coupled leading and lagging strand DNA polymerase molecules.
七种由噬菌体T4编码的蛋白质可重组出一种DNA复制装置，该装置能在体外的复制叉处催化前导链和后随链的耦合DNA合成。涉及的蛋白质包括T4 DNA聚合酶全酶（T4基因43、44/62和45的产物）、一种解螺旋（单链结合蛋白，SSB）蛋白（基因32蛋白）以及由DNA解旋酶（基因41蛋白）和引发酶（基因61蛋白）组成的T4引发体。我们在此表明，复制叉后随链上存在41蛋白可使聚合酶全酶以最大速率并具有高持续性地催化前导链DNA合成。这种前导链合成不受基因32蛋白或基因61蛋白添加的影响；41蛋白不能被dda蛋白替代，dda蛋白是另一种由T4编码的DNA解旋酶。当将61蛋白添加到41蛋白中以完成引发体时，即使在没有32蛋白的情况下，该系统中后随链上的冈崎片段合成也会伴随前导链DNA合成。然而，正如在具有耦合的前导链和后随链DNA聚合酶分子的复制叉处后随链聚合速率增加所预期的那样，添加32蛋白会减小所形成的冈崎片段的大小。
