Coupled DNA replication and nucleosome disassembly

耦合 DNA 复制和核小体分解

• 批准号: 8059931
• 负责人: JACLYN Marie JANSEN
• 金额: $ 4.84万
• 依托单位: COLD SPRING HARBOR LABORATORY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8059931
• 关键词: Animal Model; Area; Binding; Biochemical; Biological Assay; Biological Preservation; Cell Cycle Progression; Cell Nucleus; Cell division; Cell physiology; Cells; Chemicals; Chromatin; Complex; Coupled; DNA; DNA biosynthesis; DNA replication fork; Defect; Disease; Disease Progression; Drosophila melanogaster; Enzymes; Epigenetic Process; Exhibits; Failure; Flow Cytometry; Gene Expression; Genes; Goals; Growth; Histones; Human; In Vitro; Lead; Life; MCM Protein; Malignant Neoplasms; Maps; Molecular Chaperones; Mutation; Nucleosome Core Particle; Nucleosomes; Phenotype; Positioning Attribute; Pre-Replication Complex; Process; Protein Binding; Proteins; Protocols documentation; Reagent; Replication Initiation; Role; SS DNA BP; Saccharomyces cerevisiae; Saccharomycetales; Single-Stranded DNA; Stretching; Structure; System; Work; Yeasts; anticancer research; chromatin immunoprecipitation; helicase; histone modification; human disease; in vitro Assay; in vivo; insight; meter; mutant; novel; prevent; protein complex; reconstitution; research study

DESCRIPTION (provided by applicant): Compacting and organizing the meters of DNA in a human cell into the nucleus requires tight coiling into bead- like structures called nucleosomes. The nucleosomes further organize to form chromatin. Chromatin, like the DNA that surrounds it, contains information that is vital to maintaining proper gene activity. Thus, when a cell divides it is crucial for it to preserve both the sequence information contained in its DNA and the structural information in the surrounding nucleosomes. Many diseases result largely from changes in gene activity: defects in DNA replication and chromatin inheritance can lead to deleterious mutations and changes in gene expression that accelerate disease progression [28]. For example, it is hypothesized that the progression of many cancers relies on the preservation of chromatin markers over multiple cell divisions [29]. ! Despite the importance of these processes, the early steps of replication are poorly understood. In particular, compact nucleosomes must be disassembled ahead of the synthetic machinery for DNA replication to proceed, but little is known about this critical process [20]. The work outlined in this proposal will elucidate the mechanisms of chromatin unwinding during DNA replication. Specifically, it wil define the role of the replicative helicase, an enzyme that unwinds DNA, in nucleosome disassembly. Recent evidence suggests that some components of this helicase complex interact with chaperone proteins, helper proteins that bind to the core nucleosome proteins when they have dissociated from DNA [6, 13-18]. But the specific identification of a role for these proteins in nucleosome disasembly has been elusive: the chaperones have multiple functions that are intertwined with nucleosome disassembly [20]. In addition, nothing is known about the role of the full helicase complex in chromatin unwinding. Therefore, a primary goal of this proposal is to develop a novel assay using purified proteins that decouples nucleosome disassembly from other complicating cellular processes. This system will then be used to identify mutations that disrupt interactions between the helicase complex, chaperones, and core nucleosome proteins. These mutants will be a powerful reagent that will define the role of these proteins in nucleosome disassembly both in a purified system and in living cells. Specifically, these mutations should cause clear defects in DNA replication in living cells. Furthermore, they will also prevent nucleosome displacement during replication, and this can be determined by directly observing nucleosome positioning on DNA in living cells. Together, this work will provide significant insight into the mechanisms of the initiation of DNA replication and chromatin inheritance, two processes that are fundamental in our understanding of how human diseases arise. PUBLIC HEALTH RELEVANCE: This work will offer significant insight into the initiation of DNA replication and the inheritance of chromatin markers that influence gene expression: defects in either process can lead to disease. A thorough understanding of the mechanisms that control these processes will provide insight into how cells control their growth and how failures can cause disease to arise.

描述（由申请人提供）：将人类细胞中的DNA仪表压入核中，需要将其紧密地围成珠子类似于核小体的结构。核小体进一步组织形成染色质。染色质就像周围的DNA一样，包含对维持适当基因活性至关重要的信息。因此，当一个细胞分裂时，它至关重要的是要保留其DNA中包含的序列信息及其周围核小体中的结构信息。许多疾病主要是由于基因活性的变化而导致的：DNA复制和染色质遗传的缺陷会导致有害突变和基因表达的变化，从而加速了疾病进展[28]。例如，假设许多癌症的进展依赖于在多个细胞分裂上保存染色质标记[29]。呢 尽管这些过程很重要，但复制的早期步骤还是很少理解的。特别是，必须将紧凑型核小体在合成机制之前进行DNA复制之前进行拆卸，但对这个关键过程知之甚少[20]。该提案中概述的工作将阐明DNA复制过程中染色质的机理。具体而言，它将定义复制性解旋酶（一种放松DNA）在核小体拆卸中的作用。最近的证据表明，该解旋酶复合物的某些成分与伴侣蛋白，与核心核小体蛋白与DNA分离时结合的辅助蛋白，辅助蛋白[6，13-18]。但是，这些蛋白质在核小体灾难中的作用的特定鉴定是难以捉摸的：伴侣具有多种功能，这些功能与核小体拆卸相互交织在一起[20]。此外，对整个解旋酶复合物在染色质中的作用一无所知。因此，该提案的主要目标是使用纯化的蛋白质开发一种新的测定法，该蛋白质将核小体与其他复杂的细胞过程脱离。然后，该系统将用于识别破坏解旋酶复合物，伴侣和核心核小体蛋白之间相互作用的突变。这些突变体将是一种强大的试剂，它将定义这些蛋白质在纯化系统和活细胞中核糖体分解中的作用。具体而言，这些突变应导致活细胞中DNA复制的明显缺陷。此外，它们还将防止复制过程中的核小体位移，这可以通过直接观察到活细胞中DNA的核小体定位来确定。总之，这项工作将为DNA复制和染色质遗传的启动机制提供重要的见解，这是我们对人类疾病的理解的基本基础的两个过程。 公共卫生相关性：这项工作将为DNA复制的启动以及影响基因表达的染色质标记的遗传提供重大见解：两种过程中的缺陷都可以导致疾病。对控制这些过程的机制有透彻的理解将提供有关细胞如何控制其生长以及失败如何导致疾病的洞察力。