HISTONE-DNA INTERACTIONS DURING CHROMATIN BIOSYNTHESIS

申请者研究计划的长期目标是阐明 体细胞染色质生物合成的机制，并了解 将表观遗传信息传播到后代细胞。为此， 目前的建议集中在研究组蛋白-DNA相互作用 染色质的体外复制和组装。 实验将使用细胞质和核运动从 体细胞以及复制和组装因子的来源。这个 组装底物将是组蛋白耗尽的、新生的核DNA； 作为纯化的DNA质粒。在最初阶段，适当的DNA 组蛋白体外修饰的结构、状态和反应条件 将根据核酸酶升高的标准来确定组装 抗药性、核小体阶梯的产生、质粒的改变 超螺旋密度与DNA蛋白质的电泳性迁移 复合体。下一步，拓扑异构酶对组装新的 体外复制的DNA将用喜树碱和VM-26进行检测 (真核DNA拓扑异构酶I和II的特异性抑制物， )。 一旦确定了装配参数， 核心组蛋白乙酰化对组蛋白H1的沉积作用 研究了H1对体外组装的影响。作为一种 这些研究的组成部分，乙酰化水平的萌芽 含有分离的双亲组蛋白的核小体将使用 I)特异性识别乙酰化形式的组蛋白的抗血清 H4和ii)化学上可切割的生物素化核苷酸。这个 [乙酰化]H4-抗血清也将用于识别可能的核小体。 “组装因子”和组蛋白护送复合体，通过免疫转移 放射性标记的细胞提取物。使用同样的技术， 组蛋白与DNA形成核小体前组装中间体 反应也将接受测试。

The long-term objective of the applicant's research program is to elucidate the mechanisms of somatic chromatin biosynthesis, and to understand the propagation of epigenetic information to progeny cells. To this end, the present proposal focuses on studying histone-DNA interactions during chromatin replication and assembly in vitro. Experiments will be performed using cytoplasmic and nuclear exercise from somatic cells and sources of replication and assembly factors. The assembly substrate will be histone-depleted, nascent nuclear DNA< as well as purified DNA plasmids. In the initial phase, the appropriate DNA structure, histone modification state, and reaction conditions for in vitro assembly will be determined, based on the criteria of increased nuclease resistance, generation of a nucleosomal ladder, change in plasmid superhelical density, and electrophoretic migration of DNA protein complexes. Next, the topoisomerase requirements for the assembly of newly replicated DNA in vitro will be examine,d using camptothecin and VM-26 (specific inhibitors of eukaryotic DNA topoisomerases I and II, respectively). Once the parameters of assembly have been established, the influence of core histone acetylation on the deposition of histone H1 will be investigated, and the effects of H1 on assembly in vitro examined. As an integral part of these studies, the acetylation level of nascent nucleosomes containing segregated parental histones will be measured, using i) an antiserum that specifically recognizes the acetylated form of histone H4, and ii) a chemically cleavable biotinylated nucleotide. The [acetylated]H4-antiserum will also be used to identify possible nucleosome "assembly factors" and histone escort complexes, by immunopredipitating radiolabeled cell extracts. Using the same technique, the ability of histones and DNA to form pre-nucleosomal assembly intermediates in partial reactions will also be tested.