Amin A A, Hurwitz J
Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
J Biol Chem. 1992 Sep 15;267(26):18612-22.
The effect of the tus protein-terB sequence complex of Escherichia coli on the movement of the SV40 large tumor antigen (T antigen)-mediated replication fork during SV40 DNA replication in vitro has been examined. In the monopolymerase and dipolymerase systems, the tus protein-terB complex efficiently blocked the replication fork movement in a polar fashion, as observed in prokaryotic replication systems. With crude cytosolic extracts of HeLa cells, the same polarity of fork arrest was observed, but the block of replication fork movement was inefficient. These results indicate that the structure of the prokaryotic tus protein-terB complex allows it to block replication fork movement in an orientation-dependent manner. We also show that the tus protein-terB complex blocks the 3'----5' helicase action of T antigen in a polar fashion, using substrates comprised of single-stranded M13 DNA with either a 52-base pair (bp) or 29-bp duplex containing the terB sequence. The tus protein-terB complex formed on the 52-bp duplex was less effective than the complex formed on the 29-bp duplex in blocking the helicase action of T antigen. With the 52-bp duplex substrate, T antigen movement was only partially (30%) blocked by the tus protein-terB sequence complex in the active orientation, whereas the E. coli dnaB helicase moving 5'----3' was blocked more than 90% by the complex in the active orientation. However, with the shorter 29-bp duplex substrate, the complex blocked the T antigen helicase activity about 75%, whereas the dnaB helicase activity was completely blocked. Altogether, these results suggest that the T antigen helicase activity, when coupled to DNA replication, is more susceptible to arrest by the tus protein-terB complex than the T antigen functioning as a helicase alone.
已经研究了大肠杆菌的tus蛋白-terB序列复合物对体外SV40 DNA复制过程中SV40大T抗原（T抗原）介导的复制叉移动的影响。在单聚合酶和双聚合酶系统中，如在原核复制系统中观察到的那样，tus蛋白-terB复合物以极性方式有效地阻断了复制叉的移动。用HeLa细胞的粗胞质提取物时，观察到复制叉停滞的极性相同，但复制叉移动的阻断效率不高。这些结果表明，原核tus蛋白-terB复合物的结构使其能够以方向依赖的方式阻断复制叉的移动。我们还表明，使用由具有52个碱基对（bp）或包含terB序列的29-bp双链体的单链M13 DNA组成的底物，tus蛋白-terB复合物以极性方式阻断T抗原的3'→5'解旋酶作用。在52-bp双链体上形成的tus蛋白-terB复合物在阻断T抗原的解旋酶作用方面比在29-bp双链体上形成的复合物效果差。对于52-bp双链体底物，在活性方向上，T抗原的移动仅被tus蛋白-terB序列复合物部分（30%）阻断，而沿5'→3'方向移动的大肠杆菌dnaB解旋酶在活性方向上被该复合物阻断超过90%。然而，对于较短的29-bp双链体底物，该复合物阻断T抗原解旋酶活性约75%，而dnaB解旋酶活性被完全阻断。总之，这些结果表明，当与DNA复制偶联时，T抗原解旋酶活性比单独作为解旋酶发挥作用的T抗原更容易被tus蛋白-terB复合物阻断。
