DNA STRUCTURE AND DYNAMICS IN BAMHI-DNA INTERACTIONS

蛋白质对特定DNA序列的识别在许多生物学功能中起着重要作用。结构研究表明，这种特殊的识别通常涉及DNA结构的扭曲，以适应蛋白质的结合。这种特殊识别机制的两个模型已经出现。在一种情况下，蛋白质通过重新排列自身和弯曲DNA，在识别中发挥积极作用。这表明，在结合时释放的自由能足以支付结构扭曲所需的费用。然而，为了有利于复杂的形成，弯曲DNA和/或重新排列蛋白质所需的自由能必须很低。另一种模型认为DNA具有内在的灵活性，表现在局部的大振幅动力学中。这些灵活的区域提供了在蛋白质结合时容易扭曲的软点。在这两种模型中，DNA的弯曲必须是一个低自由能的过程，这表明DNA具有某种内在的灵活性。为了验证这一假设，对局部DNA动力学的研究必须伴随着结构研究。在接下来的三年里，我们计划使用高分辨率核磁共振研究和固态氘核磁共振研究来探索含有BamHI限制性内切酶结合位点的DNA十二聚体的局部结构变化和动力学。具体来说，我们提出以下方案：1)合成氘标记的DNA磷酰胺，将其掺入含有BamHI结合位点的DNA十二聚体中。2)利用固体氘核磁共振表征十二聚体中单个核苷酸的主链和糖段的局部动力学。3)利用高分辨率核磁共振提供DNA十二聚体的结构描述。

Recognition of specific DNA sequences by proteins plays an important role in many biological functions. Structural studies have shown that this specific recognition usually involves distortion of the DNA structure to accommodate protein binding. Two models for the mechanism of this specific recognition have emerged. In one, the protein plays an active role in recognition by rearranging itself and bending the DNA. It is suggested that the free energy released upon binding is sufficient to pay for that required for structural distortions. However, in order for complex formation to be favorable, the free energy required to bend the DNA and/or rearrange the protein must be low. Another model suggests that DNA possesses an intrinsic flexibility manifested in local, large amplitude dynamics. These flexible regions provide soft-spots which are easily distorted upon protein binding. In both these models, bending of the DNA must a low free energy process, suggesting that DNA has some inherent flexibility. In order to test this hypothesis, studies of local DNA dynamics must accompany structural investigations. In the next three years we plan to use high-resolution NMR studies and solid-state deuterium NMR studies to explore the local structural variations and dynamics of a DNA dodecamer containing the binding site for the BamHI restriction endonuclease. Specifically we propose the following program l) Synthesize deuterium labeled DNA phosphoramidites for incorporation into a DNA dodecamer containing the BamHI binding site. 2) Use solid-state deuterium NMR to characterize the local dynamics of backbone and sugar moieties of the individual nucleotides in the dodecamer. 3) Use high-resolution NMR to provide a structural description of the DNA dodecamer.