CHROMOSOME STRUCTURE AND GENE EXPRESSION

描述(改编自调查人员摘要)：的长期目标 这项研究是为了了解真核细胞染色体的结构域是如何 组装成调节转录的特定染色质结构。 该提案的重点是酿酒酵母的HMR基因座，该基因座是 通过一种称为沉默的机制保持转录抑制。 静音需要建立和随后维护 抑制染色质结构的限制性结构域。在HMR基因座a 沉默建立的关键一步需要Sir1蛋白 与一种被称为消音器的DNA元素有关。消音器包含 几种普遍存在的蛋白质的结合位点，包括Rap1p、Abf1p和 ORC，起源识别复合体。已经提出了这样的假设 Sir1p通过绑定到识别消音器并与其关联 沉默相关蛋白，如ORC。因为兽人被推定为 存在于所有复制起始点，而Sir1专门用于在 两个基因座，HMR和HML，Sir1被认为具有非常特殊的交互作用 和兽人在一起。 本申请中提出的实验旨在定义 Sir1p识别消音器的机制。基因筛查显示 确定在HMR消音器识别方面有缺陷的sir1srd突变体 但不是在沉默本身。这些突变体构成了基因的基础， 解决与以下几个问题相关的分子、生物化学研究 SIRP在沉默中的作用。在第一个具体目标中， 将对带有ORC的Sir1p进行调查。将在三个ORC中进行突变 基因，并测试它们抑制非交配缺陷的能力 Sir1srd等位基因。Sir1p和ORC之间的物理交互将是 用标记了Sir1p的表位进行免疫共沉淀分析。 这一分析将扩展到Sir1psrd蛋白，以确定 突变的蛋白质显示出缺陷的ORC相互作用。有没有可能 Sir1p与其他沉默结合蛋白如Rap1p和Abf1p相互作用 也将在免疫共沉淀分析中进行检查，以及是否存在 Sir1p免疫沉淀物中的其他蛋白质将被探索。 在第二个具体目标中，Sir1p的功能域是 识别消音器所需的信息将被识别。Sir1srd屏幕 将在sir1空背景中重复，以确定 Sir1p的N-末端和C-末端都包含一个 消音器识别结构域。Sir1p作为一种 消音器中的低聚物也将使用遗传和 免疫学方法。突变将在Sir1p中产生 C-末端，它包含一个假定的消音器识别结构域，以及 测试他们补充sir1srd等位基因的能力。的交互作用 带有突变的Sir1p蛋白的Sir1C末端也将通过 与表位标记蛋白的免疫共沉淀。 在第三个特定目标中，帮助Sir1p识别A的新蛋白质 消音器将使用两种不同的基因筛查进行鉴定。现有 Sir1srd等位基因的剂量抑制因子将更全面地表征 他们在沉默中扮演的角色。所需基因的功能丧失突变 Sir1p对消音器的识别将在Alpha屏幕中标识 有专门的遗传背景的人。此屏幕中识别的基因 将被分离出来，并以其对沉默的影响为特征。 ORC在DNA复制和沉默中的功能将在 第四个具体目标。HMR-E基因侧翼序列在ORC中的作用 复制和沉默的功能将通过删除来确定 分析。将HMR-E用作复制源的效率 在体内将通过二维叉迁移分析和相关的两个 ORC如何影响复制和沉默的模型。

DESCRIPTION (adapted from investigator's abstract): The long term goal of this research is to understand how domains of eukaryotic chromosomes are assembled into specific chromatin structures that regulate transcription. The proposal is focused on the HMR locus of S. cerevisiae, a locus that is kept transcriptionally repressed by a mechanism known as silencing. Silencing requires the establishment and subsequent maintenance of restricted domains of repressive chromatin structure. At the HMR locus a key step in the establishment of silencing requires that the Sir1 protein associate with a DNA element known as a silencer. The silencer contains binding sites for several ubiquitous proteins, including Rap1p, Abf1p, and ORC, the origin recognition complex. The hypothesis has been presented that Sir1p recognizes and associates with the silencer by binding to silencer-associated proteins such as ORC. Because ORC is presumed to be present at all replication origins, while Sir1 is dedicated to silencing at two loci, HMR and HML, Sir1 is postulated to have very specific interactions with ORC. The experiments proposed in this application are designed to define the mechanisms by which Sir1p recognizes a silencer. A genetic screen has identified sir1srd mutants that are defective in HMR silencer recognition but not in silencing per se. These mutants form the basis of genetic, molecular, and biochemical studies that address several issues relating to Sirp's function in silencing. In the first specific aim, the interaction of Sir1p with ORC will be investigated. Mutations will be made in three ORC genes and tested for their ability to suppress the nonmating defect of sir1srd alleles. Physical interaction between Sir1p and ORC will be examined by co-immunoprecipitation analysis using epitope tagged Sir1p. This analysis will be extended to the Sir1psrd proteins to determine whether the mutant proteins show defective ORC interactions. The possibility that Sir1p interacts with other silencer binding proteins such as Rap1p and Abf1p will also be examined in co-immunoprecipitation assays, and the presence of additional proteins in Sir1p immunoprecipitates will be probed. In the second specific aim, the functional domains of Sir1p that are required for silencer recognition will be identified. The sir1srd screen will be repeated in a sir1 null background to determine whether the N-terminus as well as the C-terminus of Sir1p contains a silencer-recognition domain. The possibility that Sir1p functions as an oligomer at silencers will also be tested using both genetic and immunological approaches. Mutations will be generated in the Sir1p C-terminus, which contains a putative silencer recognition domain, and tested for their ability to complement sir1srd alleles. Interaction of the Sir1 C-terminus with mutant Sir1p proteins will also be tested by co-immunoprecipitation with epitope tagged proteins. In the third specific aim, novel proteins that assist Sir1p to recognize a silencer will be identified using two different genetic screens. Existing dosage suppressors of sir1srd alleles will be characterized more fully for their roles in silencing. Loss-of-function mutations in genes required for Sir1p's recognition of a silencer will be identified in a screen for alpha maters in a specialized genetic background. Genes identified in this screen will be isolated and characterized for their effects on silencing. The functions of ORC in DNA replication and silencing will be examined in the fourth specific aim. The role of flanking HMR-E sequences in ORC function in replication and silencing will be determined through deletion analysis. The efficiency with which HMR-E is used as a replication origin in vivo will be tested by 2D fork migration analysis and related to two models for how ORC might affect both replication and silencing.