Clegg R M, Murchie A I, Zechel A, Carlberg C, Diekmann S, Lilley D M
Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Federal Republic of Germany.
Biochemistry. 1992 May 26;31(20):4846-56. doi: 10.1021/bi00135a016.
We have carried out fluorescence resonance energy transfer (FRET) measurements on four-way DNA junctions in order to analyze the global structure and its dependence on the concentration of several types of ions. A knowledge of the structure and its sensitivity to the solution environment is important for a full understanding of recombination events in DNA. The stereochemical arrangement of the four DNA helices that make up the four-way junction was established by a global comparison of the efficiency of FRET between donor and acceptor molecules attached pairwise in all possible permutations to the 5' termini of the duplex arms of the four-way structure. The conclusions are based upon a comparison between a series of many identical DNA molecules which have been labeled on different positions, rather than a determination of a few absolute distances. Details of the FRET analysis are presented; features of the analysis with particular relevance to DNA structures are emphasized. Three methods were employed to determine the efficiency of FRET: (1) enhancement of the acceptor fluorescence, (2) decrease of the donor quantum yield, and (3) shortening of the donor fluorescence lifetime. The FRET results indicate that the arms of the four-way junction are arranged in an antiparallel stacked X-structure when salt is added to the solution. The ion-related conformational change upon addition of salt to a solution originally at low ionic strength progresses in a continuous noncooperative manner as the ionic strength of the solution increases. The mode of ion interaction at the strand exchange site of the junction is discussed.
我们对四链 DNA 连接体进行了荧光共振能量转移（FRET）测量，以分析其整体结构及其对几种离子浓度的依赖性。了解该结构及其对溶液环境的敏感性对于全面理解 DNA 中的重组事件非常重要。通过对以所有可能排列方式成对连接到四链结构双链臂 5' 末端的供体和受体分子之间的 FRET 效率进行全局比较，确定了构成四链连接体的四条 DNA 螺旋的立体化学排列。这些结论基于对一系列在不同位置进行标记的许多相同 DNA 分子的比较，而不是对几个绝对距离的测定。文中介绍了 FRET 分析的细节；强调了与 DNA 结构特别相关的分析特征。采用了三种方法来确定 FRET 效率：（1）受体荧光增强，（2）供体量子产率降低，以及（3）供体荧光寿命缩短。FRET 结果表明，当向溶液中添加盐时，四链连接体的臂呈反平行堆叠的 X 结构排列。随着溶液离子强度的增加，向原本低离子强度的溶液中添加盐时，与离子相关的构象变化以连续的非协同方式进行。文中还讨论了连接体链交换位点处的离子相互作用模式。
