Xiao Jie, Singleton Scott F
Department of Chemistry, Rice University, P.O. Box 1892, MS 65, Houston, TX 77005, USA.
J Mol Biol. 2002 Jul 12;320(3):529-58. doi: 10.1016/s0022-2836(02)00462-x.
The RecA protein of Escherichia coli plays essential roles in homologous recombination and restarting stalled DNA replication forks. In vitro, the protein mediates DNA strand exchange between single-stranded (ssDNA) and homologous double-stranded DNA (dsDNA) molecules that serves as a model system for the in vivo processes. To date, no high-resolution structure of the key intermediate, comprised of three DNA strands simultaneously bound to a RecA filament (RecA-tsDNA complex), has been reported. We present a systematic characterization of the helical geometries of the three DNA strands of the RecA-tsDNA complex using fluorescence resonance energy transfer (FRET) under physiologically relevant solution conditions. FRET donor and acceptor dyes were used to label different DNA strands, and the interfluorophore distances were inferred from energy transfer efficiencies measured as a function of the base-pair separation between the two dyes. The energy transfer efficiencies were first measured on a control RecA-dsDNA complex, and the calculated helical parameters (h approximately 5 A, Omega(h) approximately 20 degrees ) were consistent with structural conclusions derived from electron microscopy (EM) and other classic biochemical methods. Measurements of the helical parameters for the RecA-tsDNA complex revealed that all three DNA strands adopt extended and unwound conformations similar to those of RecA-bound dsDNA. The structural data are consistent with the hypothesis that this complex is a late, post-strand-exchange intermediate with the outgoing strand shifted by about three base-pairs with respect to its registry with the incoming and complementary strands. Furthermore, the bases of the incoming and complementary strands are displaced away from the helix axis toward the minor groove of the heteroduplex, and the bases of the outgoing strand lie in the major groove of the heteroduplex. We present a model for the strand exchange intermediate in which homologous contacts preceding strand exchange arise in the minor groove of the substrate dsDNA.
大肠杆菌的RecA蛋白在同源重组和重启停滞的DNA复制叉过程中发挥着重要作用。在体外，该蛋白介导单链（ssDNA）与同源双链DNA（dsDNA）分子之间的DNA链交换，这一过程可作为体内过程的模型系统。迄今为止，尚未有关于关键中间体（由三条DNA链同时结合到RecA细丝上组成的RecA-tsDNA复合物）的高分辨率结构的报道。我们在生理相关的溶液条件下，使用荧光共振能量转移（FRET）对RecA-tsDNA复合物的三条DNA链的螺旋几何结构进行了系统表征。FRET供体和受体染料用于标记不同的DNA链，通过测量作为两种染料之间碱基对间距函数的能量转移效率来推断荧光团间的距离。首先在对照RecA-dsDNA复合物上测量能量转移效率，计算得到的螺旋参数（h约为5 Å，Ω(h)约为20°）与源自电子显微镜（EM）和其他经典生化方法的结构结论一致。对RecA-tsDNA复合物螺旋参数的测量表明，所有三条DNA链都采用与RecA结合的dsDNA相似的伸展和解旋构象。这些结构数据与以下假设一致：该复合物是链交换后的晚期中间体，其中输出链相对于输入链和互补链的对齐方式偏移了约三个碱基对。此外，输入链和互补链的碱基从螺旋轴移向异源双链体的小沟，而输出链的碱基位于异源双链体的大沟中。我们提出了一个链交换中间体的模型，其中链交换之前的同源接触发生在底物dsDNA的小沟中。
