How INO80 remodels chromatin in response to damaged DNA

INO80 如何重塑染色质以应对受损的 DNA

• 批准号: 8719735
• 负责人: Coral Yishan Zhou
• 金额: $ 3.61万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8719735
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Acetylation; Address; Affect; Biological Assay; Cancer Etiology; Cells; Characteristics; Chromatin; Chromatin Structure; Complex; Cues; DNA; DNA Damage; DNA Repair; DNA Repair Enzymes; DNA Replication Damage; DNA Sequence Rearrangement; DNA Structure; DNA repair protein; Defect; Deposition; Double Strand Break Repair; Enzymes; Fluorescence Resonance Energy Transfer; Gel; Genome; Genomic Instability; Genomics; Goals; Histones; Kinetics; Knowledge; Lead; Maintenance; Malignant Neoplasms; Measures; Methods; Mutation; Nucleosomes; Pathway interactions; Phosphorylation; Play; Process; Recruitment Activity; Research; Role; Saccharomyces cerevisiae; Signal Transduction; Site; Slide; Specificity; Step Tests; Stress; Structure; Testing; Therapeutic; Transcriptional Activation; Variant; base; cancer genome; cancer therapy; chromatin remodeling; ds-DNA; histone modification; public health relevance; reconstitution; repaired; response; restriction enzyme

DESCRIPTION (provided by applicant): Defects in a cell's ability to maintain its genomic integrity upon DNA damage and replication stress can lead to cancer. Recent evidence suggests that in addition to DNA repair enzymes, recruitment of enzymes that modify the chromatin landscape surrounding the site of damage is necessary for efficient repair. INO80 is an ATP dependent chromatin remodeling enzyme from S. cerevisiae. INO80 is recruited to sites of genomic instability, such as double strand breaks and stalled replication forks, where its enzymatic activity is required for normal kinetics of double strand break repair and replication re start. Specifically, INO80 is required for histone eviction near the break and thus recruitment of key DNA repair factors. However, how INO80 directly affects the structure of nucleosomes surrounding damaged DNA is unknown. In order to address this question, we propose to take an enzymological approach using purified enzyme and reconstituted nucleosomes to quantitatively characterize the mechanism of INO80. We hypothesize that the enzymatic activity of INO80 is directly regulated by damaged DNA through the intrinsic specificity INO80 has for nucleosomes that signal damaged DNA. To test this hypothesis, we will use a combination of FRET based, gel based, and restriction enzyme based methods to quantify the kinetics of both fast steps and slow steps of nucleosome remodeling and test how the steps of remodeling are changed in the presence of nucleosome substrates that specific to damaged DNA. In Aim 1, we will investigate the effect that broken DNA ends or single stranded overhangs have on the remodeling and ATPase activity of INO80, and in Aim 2, we will investigate the effect of damage- specific histone modifications on the same activities. Understanding how INO80 responds to these damage- specific nucleosomes will further our understanding of how the chromatin landscape must be actively altered under conditions of genomic instability in order to facilitate repair of damaged DNA. This knowledge will useful in cancer therapies that target chromatin remodeling specifically under conditions of genomic instability.

描述（由申请人提供）：细胞在 DNA 损伤和复制压力下维持其基因组完整性的能力缺陷可能导致癌症。最近的证据表明，除了 DNA 修复酶之外，招募改变损伤部位周围染色质景观的酶对于有效修复也是必要的。 INO80 是一种来自酿酒酵母的 ATP 依赖性染色质重塑酶。 INO80 被招募到基因组不稳定位点，例如双链断裂和停滞的复制叉，其中其酶活性是双链断裂修复和复制再复制的正常动力学所必需的。 开始。具体来说，INO80 是断裂附近的组蛋白驱逐所必需的，从而招募关键的 DNA 修复因子。然而，INO80 如何直接影响受损 DNA 周围的核小体结构尚不清楚。为了解决这个问题，我们建议采用酶学方法，使用纯化的酶和重构的核小体来定量表征 INO80 的机制。我们假设 INO80 的酶活性通过 INO80 对发出受损 DNA 信号的核小体的内在特异性直接受到受损 DNA 的调节。为了检验这一假设，我们将结合使用基于 FRET、基于凝胶和基于限制性酶的方法来量化核小体重塑的快步和慢步的动力学，并测试在核小体存在的情况下重构步骤如何改变。特定于受损 DNA 的底物。在目标 1 中，我们将研究断裂的 DNA 末端或单链突出端对 INO80 的重塑和 ATP 酶活性的影响，在目标 2 中，我们将研究损伤特异性组蛋白修饰对相同活性的影响。了解 INO80 如何响应这些损伤特异性核小体将进一步加深我们对在基因组不稳定条件下染色质景观必须如何主动改变以促进受损 DNA 修复的理解。这些知识将有助于在基因组不稳定条件下针对染色质重塑的癌症治疗。