How INO80 remodels chromatin in response to damaged DNA

INO80 如何重塑染色质以应对受损的 DNA

基本信息

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
项目类别：
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

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

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项目摘要

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损伤和复制应力上保持其基因组完整性的能力的缺陷会导致癌症。最近的证据表明，除了DNA修复酶外，还需要募集围绕损伤部位的染色质景观的酶，对于有效的维修也是必需的。 INO80是酿酒酵母的依赖性ATP染色质重塑酶。 INO80被招募到基因组不稳定性部位，例如双链断裂和停滞的复制叉，在这种酶活性的酶促活性需要双链断裂修复和复制的正常动力学需要开始。具体而言，INO80在破裂附近的组蛋白驱逐并因此募集了关键DNA修复因子所必需。但是，Ino80如何直接影响受损DNA周围核小体的结构是未知的。为了解决这个问题，我们建议使用纯化的酶和重构的核小体采用酶学方法，以定量地表征INO80的机制。我们假设INO80的酶促活性通过固有特异性INO80 hAS直接受到DNA的调节，对信号损坏DNA的核小体。为了检验这一假设，我们将使用基于FRET的，基于FRET的，基于凝胶和限制酶的方法的组合来量化核小体重塑的快速步骤和慢速步骤的动力学，并测试在特定于受损DNA的特定核小组底物的情况下，在存在核小体底物的情况下，重塑步骤如何更改。在AIM 1中，我们将研究损坏的DNA末端或单链悬垂物对INO80的重塑和ATPase活性具有破裂的作用，在AIM 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如何应对这些损伤特异性核小体将进一步了解我们对在基因组不稳定性条件下必须积极改变染色质景观的理解，以促进修复受损的DNA。这些知识将在基因组不稳定性条件下专门针对染色质重塑的癌症疗法有用。