BIOPHYSICAL STUDIES OF OLIGOPURINE TRACTS

低聚嘌呤序列在体外已被证明是多功能元素， 能够假设几种不同的DNA构象。一个 最近对已发表的DNA中超过50万个核苷酸的搜索 序列已经表明，超过10个碱基对的长链 连续的嘌呤残留物在很大程度上并一直被高估 真核生物，但不是原核生物。这一假说激发了 这项提案中提出的工作是，这种长的寡尿路具有 不同于散装DNA的物理特征，以及 真核细胞可以利用这些特性来发挥 生物效应。拟议研究的目的是为了了解 低聚嘌呤的物理和生物特性- 聚合体DNA中的寡嘧啶[(Pu-Py)n]区。 该提案首先对一个化合物的圆二向色性光谱进行了调查 不同溶液下的已定义序列多脱氧核苷酸数量 条件，以便掌握起点在哪里--即在什么时候 (Pu-Py)n区域的构象与块体明显不同吗 DNA然后，调查的路线继续进行量化 确定可能很重要的序列的性质 它们在体内扮演的角色：它们影响核小体的能力 队形。这将通过竞争性核小体重组来完成。 由我们实验室开发的一种化验方法，它允许测定 相互竞争的核小体形成自由能的差异 多核苷酸。合作的瞬变电双折射研究， 测量合成聚合物的弯曲硬度，然后将 进行并与CD研究中所见的结构变化相关 核小体重组分析中所见的功能变化。

Oligopurine sequences have been shown to be versatile elements in vitro, capable of assuming several different alternative DNA conformations. A recent search of over one half million nucleotides of published DNA sequences has shown that long tracts greater than 10 base pairs of contiguous purine residues are greatly and consistently overrepresented in eukaryotic, but not prokaryotic, organisms. The hypothesis motivating the work presented in this proposal is that such long oligopurine tracts have physical characteristics that are different from bulk DNA, and that the eukaryotic cell can take advantage of these characteristics to exert biological effects. The purpose of the proposed research is to understand the physical and, to an extent, the biological properties of oligopurine- oligopyrimidine [(Pu-Py)n] regions in polymeric DNA. The proposal begins with a survey of the circular dichroism spectra of a number of defined-sequence polydeoxynucleotides under a variety of solution conditions in order to grasp where the beginning is, -i.e., at what point are (Pu-Py)n regions in a recognizably different conformation than bulk DNA. The line of investigation then continues with the quantitative determination of a property of the sequences that is likely to be important to the role they play in vivo: their ability to affect nucleosome formation. This will be done using a competitive nucleosome reconstitution assay, developed by our laboratory, that allows the determination of the difference in the free energy of nucleosome formation between competing polynucleotides. Collaborative transient electric birefringence studies, measuring the bending stiffness of the synthetic polymers, will then be performed and correlated to the structural changes seen in CD studies and the functional changes seen in nucleosome reconstitution assays.