Burke R L, Alberts B M, Hosoda J
J Biol Chem. 1980 Dec 10;255(23):11484-93.
The proteolytic removal of about 60 amino acids from the COOH terminus of the bacteriophage T4 helix-destabilizing protein (gene 32 protein) produces 32I, a 27,000-dalton fragment which still binds tightly and cooperatively to single-stranded DNA. The substitution of 32I protein for intact 32 protein in the seven-protein T4 replication complex results in dramatic changes in some of the reactions catalyzed by this in vitro DNA replication system, while leaving others largely unperturbed. 1. Like intact 32 protein, the 32I protein promotes DNA synthesis by the DNA polymerase when the T4 polymerase accessory proteins (gene 44/62 and 45 proteins) are also present. The host helix-destabilizing protein (Escherichia coli ssb protein) cannot replace the 32I protein for this synthesis. 2. Unlike intact 32 protein, 32I protein strongly inhibits DNA synthesis catalyzed by the T4 DNA polymerase alone on a primed single-stranded DNA template. 3. Unlike intact 32 protein, the 32I protein strongly inhibits RNA primer synthesis catalyzed by the T4 gene 41 and 61 proteins and also reduces the efficiency of RNA primer utilization. As a result, de novo DNA chain starts are blocked completely in the complete T4 replication system, and no lagging strand DNA synthesis occurs. 4. The 32I protein does not bind to either the T4 DNA polymerase or to the T4 gene 61 protein in the absence of DNA; these associations (detected with intact 32 protein) would therefore appear to be essential for the normal control of 32 protein activity, and to account at least in part for observations 2 and 3, above. We propose that the COOH-terminal domain of intact 32 protein functions to guide its interactions with the T4 DNA polymerase and the T4 gene 61 RNA-priming protein. When this domain is removed, as in 32*I protein, the helix destabilization induced by the protein is controlled inadequately, so that polymerizing enzymes tend to be displaced from the growing 3'-OH end of a polynucleotide chain and are thereby inhibited. Eukaryotic helix-destabilizing proteins may also have similar functional domains essential for the control of their activities.
从噬菌体T4解螺旋蛋白（基因32蛋白）的COOH末端蛋白水解去除约60个氨基酸，产生32I，一个27,000道尔顿的片段，它仍然紧密且协同地结合单链DNA。在七蛋白T4复制复合物中用32I蛋白替代完整的32蛋白，会导致这个体外DNA复制系统催化的一些反应发生显著变化，而其他反应基本不受影响。1. 与完整的32蛋白一样，当T4聚合酶辅助蛋白（基因44/62和45蛋白）也存在时，32I蛋白可促进DNA聚合酶进行DNA合成。宿主解螺旋蛋白（大肠杆菌单链结合蛋白）不能替代32I蛋白进行这种合成。2. 与完整的32蛋白不同，32I蛋白强烈抑制T4 DNA聚合酶单独在带引物的单链DNA模板上催化的DNA合成。3. 与完整的32蛋白不同，32I蛋白强烈抑制T4基因41和61蛋白催化的RNA引物合成，还降低RNA引物的利用效率。结果，在完整的T4复制系统中从头开始的DNA链起始被完全阻断，并且没有滞后链DNA合成发生。4. 在没有DNA的情况下，32I蛋白不与T4 DNA聚合酶或T4基因61蛋白结合；因此，这些结合（用完整的32蛋白检测到）对于32蛋白活性的正常调控似乎至关重要，并且至少部分解释了上述观察结果2和3。我们提出，完整的32蛋白的COOH末端结构域的功能是指导其与T4 DNA聚合酶和T4基因61 RNA引发蛋白的相互作用。当这个结构域被去除时，如在32I蛋白中，由该蛋白诱导的螺旋解稳定作用控制不足，使得聚合酶倾向于从多核苷酸链不断增长的3'-OH末端被取代，从而受到抑制。真核解螺旋蛋白可能也有类似的对其活性控制至关重要的功能结构域。
