The 2B domain of the Escherichia coli Rep protein is not required for DNA helicase activity
The 2B domain of the Escherichia coli Rep protein is not required for DNA helicase activity
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DOI：
10.1073/pnas.242479399
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发表时间：
2002-12-10
影响因子：
11.1
通讯作者：
Lohman, TM
中科院分区：
文献类型：
--
作者：
Cheng, W;Brendza, KM;Lohman, TM
大肠杆菌Rep蛋白是噬菌体phiX174在大肠杆菌中复制所必需的3‘-5’SH DNA解旋酶，在结构上与大肠杆菌UvrD解旋酶和嗜热脂肪芽孢杆菌PcrA解旋酶同源。以前对Rep蛋白与单链DNA结合的结晶学研究表明，它可以经历一个很大的构象变化，包括它的2B亚结构域围绕连接到2A亚结构域的铰链区旋转约130度。基于对PcrA的结晶学研究，其2B亚区被认为是其双链DNA结合位点的一部分，并在双链失稳中发挥作用。为了测试2B亚域在Rep催化的双链DNA解离中的作用，我们删除了它的2B亚域，用三个甘氨酸取代它，形成了RepDelta2B蛋白。这种RepDelta2B蛋白可以支持phiX174在rep(-)大肠杆菌菌株中的复制，尽管含有rep Delta2B基因的大肠杆菌的生长速度大约是野生型rep基因的1.5倍。预稳态单周转DNA解离动力学实验表明，纯化的RepDelta2B蛋白在体外具有DNA解旋酶活性，解离18个碱基的DNA双链的速度至少与野生型Rep一样快，解离程度和对DNA底物的亲和力更高。这些研究表明，在体内或体外，Rep的2B结构域不是DNA解旋酶活性所必需的，而且它不利于DNA在体外的解离。
The Escherichia coli Rep protein is a 3' to 5' SH DNA helicase required for replication of bacteriophage phiX174 in E. coh, and is structurally homologous to the E. coli UvrD helicase and the Bacillus stearothermophilus PcrA helicase. Previous crystallographic studies of Rep protein bound to single-stranded DNA revealed that it can undergo a large conformational change consisting of an approximate to130degrees rotation of its 2B subdomain about a hinge region connected to the 2A subdomain. Based on crystallographic studies of PcrA, its 2B subdomain has been proposed to form part of its duplex DNA binding site and to play a role in duplex destabilization. To test the role of the 2B subdomain in Rep-catalyzed duplex DNA unwinding, we have deleted its 2B subdomain, replacing it with three glycines, to form the RepDelta2B protein. This RepDelta2B protein can support phiX174 replication in a rep(-) E. coli strain, although the growth rate of E. coli containing the repDelta2B gene is approximate to1.5-fold slower than with the wild-type rep gene. Pre-steady-state, single-turnover DNA unwinding kinetics experiments show that purified RepDelta2B protein has DNA helicase activity in vitro and unwinds an 18-bp DNA duplex with rates at least as fast as wild-type Rep, and with higher extents of unwinding and higher affinity for the DNA substrate. These studies show that the 2B domain of Rep is not required for DNA helicase activity in vivo or in vitro, and that it does not facilitate DNA unwinding in vitro.