Li X, Wang H, Seeman N C
Department of Chemistry, New York University, New York 10003, USA.
Biochemistry. 1997 Apr 8;36(14):4240-7. doi: 10.1021/bi9629586.
The Holliday junction is a key intermediate in genetic recombination. This is a four-stranded branched DNA structure, whose double-helical arms are stacked in two domains; two of the strands are roughly helical, and the other two cross over between domains. Switching the strands between these two roles is known as crossover isomerization; this postulated reversal is thought to be one of the key transformations that the Holliday junction can undergo, because it can lead to changing the products from patch to splice recombinants. We present direct evidence that this reaction can indeed occur in Holliday junctions in solution. We have constructed a double-crossover molecule containing a branched junction, constrained not to be in its favored conformation. This junction is released from the double-crossover molecule by digestion with restriction endonucleases. We demonstrate by means of hydroxyl radical autofootprinting that the junction changes its crossover isomer spontaneously when released from the double crossover. We control for the possibility that the experimental protocol causes the isomerization. We also exclude dissociation and interaction with other molecules in solution as contributing to the phenomenon. Thus, crossover isomerization is an authentic spontaneous transformation of Holliday junctions.
霍利迪连接体是基因重组中的关键中间体。这是一种四链分支DNA结构，其双螺旋臂堆叠在两个结构域中；其中两条链大致呈螺旋状，另外两条链在结构域之间交叉。在这两种作用之间切换链被称为交叉异构化；这种假定的反转被认为是霍利迪连接体能够经历的关键转变之一，因为它可以导致产物从拼接型重组体转变为剪接型重组体。我们提供了直接证据表明这种反应确实可以在溶液中的霍利迪连接体中发生。我们构建了一个包含分支连接体的双交叉分子，该连接体被限制在非其偏好的构象中。通过用限制性内切核酸酶消化，该连接体从双交叉分子中释放出来。我们通过羟基自由基自足迹法证明，当从双交叉结构中释放时，连接体会自发改变其交叉异构体。我们控制了实验方案导致异构化的可能性。我们还排除了解离以及与溶液中其他分子的相互作用导致该现象的可能性。因此，交叉异构化是霍利迪连接体真实的自发转变。
