PHYSICAL CHEMISTRY OF NUCLEIC ACIDS

这里提出的研究的目的是为了阐明 基本染色质纤维采用的有序结构作为 体外离子强度，并阐明相关的分子因素 为了这些结构的稳定性。这些研究是继续进行的 我们之前在染色质结构上所做的努力。 染色质纤维将在空气中用扫描力显微镜成像 (SFM)以：i)表征盐的中间形式。 缩合过程；II)研究连接子组蛋白HI的作用 和H5及其球状和尾状结构域上的完整性和稳定性 III)阐明了组蛋白乙酰化对 纤维结构；以及iv)实时跟踪盐诱导的 使用新开发的液体的冷凝工艺。运行 SFM的能力。 连接子组蛋白H1和H5在细胞的进出角中的作用 核小体周围的连接物DNA将用 不同离子条件下的三核小体和H_1超螺旋DNA复合体 强项。HI的各个领域的参与将是 阐述了部分胰酶蛋白水解物对 这些系统的结构参数。同时使用SFM和沉淀法 速度，我们还将表征弯曲和 盐诱导缩合过程中连接体DNA的折叠 二核小体。 结构研究将与力学研究相辅相成 测定盐诱导凝聚的能量学。我们将绘制 不同离子强度下的核小体间净相互作用势 使用我们实验室开发的单分子操作方法。 上述研究的结果将用于计算机 纤维冷凝中间产物的模拟。这些模拟 将结合：i)连接物的离子强度依赖性 刚性；ii)进出角的离子强度依赖性 核小体周围连接子的分布；III)随机性 连接子的可变性；以及iv)相互作用的潜力 核小体在给定的离子强度下。结构的组合， 机械和模拟研究将提供关于 影响纤维结构稳定性的分子因素。

The objectives of the studies proposed here are to elucidate the higher order structures adopted by the basic chromatin fiber as a function of ionic strength in vitro, and elucidate the molecular factors responsible for the stability of these structures. These studies are a continuation of our previous efforts on chromatin structure. Chromatin fibers will be imaged in air with the Scanning Force Microscope (SFM) to: i) characterize intermediate forms of the salt.induced condensation process; ii) investigate the role of the linker histones HI and H5 and their globular and tail domains on the integrity and stability of the fiber; iii) elucidate the consequences of histone acetylation on the fiber structure; and iv) follow in real time the salt-induced condensation process using the newly.developed liquid.operating capability of the SFM. The role of the linker histones H1 and H5 on the entry and exit angle of the linker DNA around the nucleosome will be investigated using trinucleosomes and H1-supercoiled DNA complexes at various ionic strengths. The participation of the various domains of HI will be addressed following the effects of partial trypsin proteolysis on the structural parameters of these systems. Using both SFM and sedimentation velocity, we will also characterize the contribution of bending and folding of the linker DNA during the salt-induced condensation process of dinucleosomes. The structural studies will be complemented with mechanical studies to determine the energetics of salt-induced condensation. We will map the net internucleosomal interaction potential at various ionic strengths using single molecule manipulation methods developed in our laboratory. The results from the above studies will be used to perform computer simulations of condensation intermediates of the fiber. These simulations will incorporate: i) the ionic strength dependence of the linker rigidity; ii) the ionic strength dependence of the entry and exit angle distributions of the linker around the nucleosome; iii) the random variability of linkers; and iv) the interaction potential among nucleosomes at a given ionic strength. The combination of structural, mechanical and simulation studies will provide insights about the molecular factors responsible for the stability of the fiber structure.