通过单周转化学淬灭流动动力学研究确定大肠杆菌RecBCD解旋酶的DNA解旋步长。

DNA unwinding step-size of E. coli RecBCD helicase determined from single turnover chemical quenched-flow kinetic studies.

作者信息

Lucius Aaron L, Vindigni Alessandro, Gregorian Razmic, Ali Janid A, Taylor Andrew F, Smith Gerald R, Lohman Timothy M

机构信息

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 S. Euclid Ave., Box 8231, St. Louis, MO 63110, USA.

出版信息

J Mol Biol. 2002 Nov 29;324(3):409-28. doi: 10.1016/s0022-2836(02)01067-7.

DOI:10.1016/s0022-2836(02)01067-7

PMID:12445778

Abstract

The mechanism by which Escherichia coli RecBCD DNA helicase unwinds duplex DNA was examined in vitro using pre-steady-state chemical quenched-flow kinetic methods. Single turnover DNA unwinding experiments were performed by addition of ATP to RecBCD that was pre-bound to a series of DNA substrates containing duplex DNA regions ranging from 24 bp to 60 bp. In each case, the time-course for formation of completely unwound DNA displayed a distinct lag phase that increased with duplex length, reflecting the transient formation of partially unwound DNA intermediates during unwinding catalyzed by RecBCD. Quantitative analysis of five independent sets of DNA unwinding time courses indicates that RecBCD unwinds duplex DNA in discrete steps, with an average unwinding "step-size", m=3.9(+/-1.3)bp step(-1), with an average unwinding rate of k(U)=196(+/-77)steps s(-1) (mk(U)=790(+/-23)bps(-1)) at 25.0 degrees C (10mM MgCl(2), 30 mM NaCl (pH 7.0), 5% (v/v) glycerol). However, additional steps, not linked directly to DNA unwinding are also detected. This kinetic DNA unwinding step-size is similar to that determined for the E.coli UvrD helicase, suggesting that these two SF1 superfamily helicases may share similar mechanisms of DNA unwinding.

摘要

利用预稳态化学淬灭流动动力学方法在体外研究了大肠杆菌RecBCD DNA解旋酶解开双链DNA的机制。通过向预先结合到一系列含有24 bp至60 bp双链DNA区域的DNA底物上的RecBCD添加ATP来进行单轮DNA解旋实验。在每种情况下，完全解开的DNA形成的时间进程都显示出一个明显的滞后阶段，该阶段随着双链长度的增加而增加，这反映了在RecBCD催化的解旋过程中部分解开的DNA中间体的瞬时形成。对五组独立的DNA解旋时间进程进行定量分析表明，RecBCD以离散步骤解开双链DNA，平均解旋“步长”m = 3.9（±1.3）bp步⁻¹，在25.0℃（10 mM MgCl₂，30 mM NaCl（pH 7.0），5%（v/v）甘油）下平均解旋速率k(U)=196（±77）步 s⁻¹（mk(U)=790（±23）bps⁻¹）。然而，还检测到了与DNA解旋没有直接联系的其他步骤。这种动力学DNA解旋步长与为大肠杆菌UvrD解旋酶确定的步长相似，这表明这两种SF1超家族解旋酶可能具有相似的DNA解旋机制。