McKinney Sean A, Déclais Anne-Cécile, Lilley David M J, Ha Taekjip
Physics department, University of Illinois, Urbana-Champaign, 61801, USA.
Nat Struct Biol. 2003 Feb;10(2):93-7. doi: 10.1038/nsb883.
The four-way DNA (Holliday) junction is the central intermediate of genetic recombination, but the dynamic aspects of this important structure are presently unclear. Although transitions between alternative stacking conformers have been predicted, conventional kinetic studies are precluded by the inability to synchronize the junction in a single conformer in bulk solution. Using single-molecule fluorescence methodology we have been able to detect these transitions. The sequence dependence, the influence of counterions and measured energetic barriers indicate that the conformer transition and branch migration processes share the unstacked, open structure as the common intermediate but have different rate-limiting steps. Relative rates indicate that multiple conformer transitions occur at each intermediate step of branch migration, allowing the junction to reach conformational equilibrium. This provides a mechanism whereby the sequence-dependent conformational bias could determine the extent of genetic exchange upon junction resolution.
四链DNA（霍利迪）连接体是基因重组的核心中间体，但目前这一重要结构的动态特性尚不清楚。尽管已预测到交替堆积构象之间会发生转变，但由于无法在本体溶液中将连接体同步到单一构象，传统的动力学研究受到了限制。利用单分子荧光方法，我们得以检测到这些转变。序列依赖性、抗衡离子的影响以及测得的能垒表明，构象转变和分支迁移过程共享未堆积的开放结构作为共同中间体，但具有不同的限速步骤。相对速率表明，在分支迁移的每个中间步骤都会发生多个构象转变，使连接体达到构象平衡。这提供了一种机制，通过该机制序列依赖性的构象偏向可在连接体拆分时决定基因交换的程度。
