Sikora Bartek, Eoff Robert L, Matson Steven W, Raney Kevin D
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
J Biol Chem. 2006 Nov 24;281(47):36110-6. doi: 10.1074/jbc.M604412200. Epub 2006 Sep 19.
The F plasmid TraI protein (DNA helicase I) plays an essential role in conjugative DNA transfer as both a transesterase and a helicase. Previous work has shown that the 192-kDa TraI protein is a highly processive helicase, catalytically separating >850 bp under steady-state conditions. In this report, we examine the kinetic mechanism describing DNA unwinding of TraI. The kinetic step size of TraI was measured under both single turnover and pre-steady-state conditions. The resulting kinetic step-size estimate was approximately 6-8 bp step(-1). TraI can separate double-stranded DNA at a rate of approximately 1100 bp s(-1), similar to the measured unwinding rate of the RecBCD helicase, and appears to dissociate very slowly from the 3' terminus following translocation and strand-separation events. Analyses of pre-steady-state burst amplitudes indicate that TraI can function as a monomer, similar to the bacteriophage T4 helicase, Dda. However, unlike Dda, TraI is a highly processive monomeric helicase, making it unique among the DNA helicases characterized thus far.
F质粒TraI蛋白（DNA解旋酶I）作为转酯酶和解旋酶，在接合性DNA转移中发挥着至关重要的作用。先前的研究表明，192 kDa的TraI蛋白是一种高度持续的解旋酶，在稳态条件下能催化分离超过850 bp的DNA。在本报告中，我们研究了描述TraI解开DNA的动力学机制。在单周转和预稳态条件下测量了TraI的动力学步长。所得的动力学步长估计约为6 - 8 bp步长（-1）。TraI能以约1100 bp s（-1）的速率分离双链DNA，这与测得的RecBCD解旋酶的解旋速率相似，并且在移位和链分离事件后似乎从3'末端解离得非常缓慢。对预稳态爆发幅度的分析表明，TraI可以作为单体发挥作用，类似于噬菌体T4解旋酶Dda。然而，与Dda不同的是，TraI是一种高度持续的单体解旋酶，这使其在迄今为止已表征的DNA解旋酶中独一无二。
