Real-time observation of bacteriophage T4 gp41 helicase reveals an unwinding mechanism
Real-time observation of bacteriophage T4 gp41 helicase reveals an unwinding mechanism
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DOI：
10.1073/pnas.0709793104
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发表时间：
2007-12-11
影响因子：
11.1
通讯作者：
Croquette, Vincent
中科院分区：
文献类型：
--
作者：
Lionnet, Timothee;Spiering, Michelle M.;Croquette, Vincent
解旋酶是一种将ATP水解酶与双链(DS)核酸解链相结合的酶。噬菌体T4解旋酶(Gp41)是一种六聚体解旋酶，它促进DNA在一个被称为复制体的高度协调的蛋白质复合体中复制。尽管最近取得了进展，但gp41的解离机制和复制体内的调控相互作用仍不清楚。在这里，我们使用单个系链DNA发夹作为gp41介导的dsDNA解离和3碱基对(BP)分辨率的单链(Ss)DNA易位的实时报告。虽然gp41在单链DNA上的移位速度与体内复制叉一样快(约为400bp/S)，但其解离速度外推至零力要慢得多(约为30bp/S)。总而言之，我们的结果有两个含义：第一，gp41通过一种被动的机制解开DNA；第二，这种弱的解旋酶不能单独有效地解开T4基因组。我们的结果表明，重要的调控发生在复制体内，以实现快速和渐进的复制。
Helicases are enzymes that couple ATP hydrolysis to the unwinding of double-stranded (ds) nucleic acids. The bacteriophage T4 helicase (gp41) is a hexameric helicase that promotes DNA replication within a highly coordinated protein complex termed the replisome. Despite recent progress, the gp41 unwinding mechanism and regulatory interactions within the replisome remain unclear. Here we use a single tethered DNA hairpin as a real-time reporter of gp41-mediated dsDNA unwinding and single-stranded (ss) DNA translocation with 3-base pair (bp) resolution. Although gp41 translocates on ssDNA as fast as the in vivo replication fork (approximate to 400 bp/s), its unwinding rate extrapolated to zero force is much slower (approximate to 30 bp/s). Together, our results have two implications: first, gp41 unwinds DNA through a passive mechanism; second, this weak helicase cannot efficiently unwind the T4 genome alone. Our results suggest that important regulations occur within the replisome to achieve rapid and processive replication.