HISTONE-DNA INTERACTIONS DURING CHROMATIN BIOSYNTHESIS

申请人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 表观遗传信息向子代细胞的传播。 为此中央 目前的建议侧重于研究组蛋白-DNA相互作用， 体外染色质复制和组装。 实验将使用细胞质和细胞核运动进行， 体细胞以及复制和组装因子的来源。 的 组装底物将是组蛋白耗尽的新生核DNA， 作为纯化的DNA质粒。 在最初阶段，适当的DNA 结构、组蛋白修饰状态和体外反应条件 将基于核酸酶增加的标准来确定组装 耐药性，核小体梯状体的产生，质粒的变化 超螺旋密度与DNA蛋白质电泳迁移 配合物 接下来，新的组装的拓扑异构酶要求 用喜树碱和VM-26检测体外复制的DNA （真核DNA拓扑异构酶I和II的特异性抑制剂， 分别）。 一旦装配参数确定， 核心组蛋白乙酰化对组蛋白H1的沉积将是 研究，并在体外检测H1对组装的影响。 作为 这些研究的组成部分，乙酰化水平的新生 将测量含有分离的亲本组蛋白的核小体，使用 i）特异性识别组蛋白乙酰化形式的抗血清， H4，和ii）可化学裂解的生物素化核苷酸。 的 [乙酰化]H4-抗血清也将用于鉴定可能的核小体 “组装因子”和组蛋白护送复合物，通过免疫沉淀， 放射性标记的细胞提取物。 使用同样的技术， 组蛋白和DNA形成部分核小体前组装中间体 反应也将被测试。

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.