Role of Chromatin and ATP-dependent Remodeling on DNA DSB Processing

染色质和 ATP 依赖性重塑对 DNA DSB 加工的作用

• 批准号: 8218053
• 负责人: Nicholas Leonard Adkins
• 金额: $ 4.11万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-02 至 2012-11-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8218053
• 关键词: Address; Affect; Alleles; Binding Sites; Biochemical; Biochemical Genetics; Biological Assay; Cancer Etiology; Cell physiology; Chemicals; Chimeric Proteins; Chromatin; Chromatin Remodeling Factor; Chromatin Structure; Chromatin Structure Alteration; Chromosomal Rearrangement; Chromosome Structures; Critical Pathways; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA replication fork; Defect; Dependence; Development; Disease; Double Strand Break Repair; Early Diagnosis; Engineering; Environment; Enzymes; Excision; Expeditions; Fungal Genome; Genes; Genetic; Genome Stability; Genomics; Goals; Histones; Human; Immunoblotting; In Vitro; Investigation; Ionizing radiation; Lead; Light; Maintenance; Meiosis; Methods; Molecular Genetics; Monitor; Mutation; Nucleosomes; Pathway interactions; Patients; Phosphorylation; Predisposition; Process; Reactive Oxygen Species; Research; Role; Single-Stranded DNA; Source; Structure; System; Techniques; Variant; Yeasts; age related; cancer therapy; cancer type; chromatin modification; chromatin remodeling; design; environmental agent; homologous recombination; human H2AX protein; in vivo; mutant; novel; programs; protein protein interaction; public health relevance; reconstitution; repaired; theories

DESCRIPTION (provided by applicant): Role of Chromatin and ATP-dependent Remodeling on DNA Double-Strand Break Processing The repair of DNA double strand breaks (DSBs) is known to be a critical process in the maintenance of genomic integrity. DSBs can arise from both environmental agents (ionizing radiation, mutagenic chemicals, etc.) and/or endogenous sources (oxygen radicals, collapsed DNA replication forks, or programmed cellular processes such as meiosis). Defects in this repair pathway can lead to mutations such as gene translocations and gross chromosomal rearrangements which can cause cancer. While recent studies have shed light on some of the processes of DSB repair, the exact mechanism has yet to be elucidated. Furthermore, despite characterization of processing enzymes involved in DSB repair, the role of chromatin in this process is currently ill-defined. One prevalent theory suggests that the loss of histones and/or nucleosomes is required for the processing of DSBs. In support of this theory, previous studies have indicated that recruitment of Ino80, a chromatin remodeling complex, is required for proper DSB repair. Ino80 recruitment is dependent on histone H2AX phosphorylation and is believed to facilitate DSB repair through possible alteration of the structure of chromatin adjacent to breaks. Our preliminary results indicate that Ino80 recruitment itself may be dependent on DSB processing. This project will examine the genetic requirements to Ino80 recruitment to an induced DSB at a single specific locus. This analysis will be performed in the presence and absence of key processing enzymes to more clearly delineate the requirements for Ino80 recruitment. To determine the effect of chromatin on DSB repair, an in vitro system using purified DSB processing enzymes will be employed. This recently-developed biochemical assay allows for revelation of the minimum enzymatic components necessary for resection of DNA in vitro. While previous assays achieved successful resection with purified processing enzymes, only naked DNA was used as a substrate. This newly developed resection assay will be utilized to examine nucleosomal templates, which are more reflective of the environment of genomic DNA in vivo. This investigation is designed to shed light on the genetic and molecular mechanism of DNA DSB repair, specifically the role of chromatin and chromatin remodeling enzymes. Specifically it will ask: What is the effect of nucleosomes on DSB resection and what enzymatic processes are required to overcome this barrier? By identifying the enzymatic processes and order of recruitment required to overcome the barrier of chromatin, the understanding of DSB repair will be furthered. This characterization of chromatin and ATP- dependent remodelers during DSB repair will allow expedition of the development of pharmacologic agents for the treatment of cancer by possibly targeting chromatin remodeling at DSBs to promote repair through homologous recombination. PUBLIC HEALTH RELEVANCE: This proposal describes research that is focused on how chromosome structure affects repair of DNA breaks, and how the normal cellular machinery affects this structure, influencing DNA repair. Specifically, we propose studies on chromatin during DNA resection which is required for homologous recombination. Repair of DNA through homologous recombination is one of the critical pathways in the maintenance of genomic integrity which has been shown to influence several pathological human disorders such as age related diseases and certain types of cancer.

描述(申请人提供)：染色质和依赖于ATP的重塑在DNA双链断裂过程中的作用DNA双链断裂(DSB)的修复被认为是维持基因组完整性的关键过程。DSB可由两种环境因素(电离辐射、致突变化学品等)引起。和/或内源性来源(氧自由基、倒塌的DNA复制叉或程序化的细胞过程，如减数分裂)。这种修复途径的缺陷可能会导致突变，如基因易位和大染色体重排，从而导致癌症。虽然最近的研究已经阐明了DSB修复的一些过程，但确切的机制尚未阐明。此外，尽管对参与DSB修复的加工酶进行了表征，但染色质在这一过程中的作用目前还不清楚。一种流行的理论认为，组蛋白和/或核小体的丢失是处理DSB所必需的。为了支持这一理论，先前的研究表明，适当的DSB修复需要重新招募染色质重塑复合体INO80。INO80的重新募集依赖于组蛋白H2AX的磷酸化，并被认为通过可能改变断裂附近的染色质结构来促进DSB修复。我们的初步结果表明，INO80的招募本身可能依赖于DSB的加工。该项目将研究INO80在单个特定基因座上对诱导DSB的重新招募的遗传要求。这项分析将在关键加工酶存在和不存在的情况下进行，以更清楚地描述INO80招募的要求。为了确定染色质在DSB修复中的作用，将使用纯化的DSB处理酶的体外系统。这种最近开发的生化分析方法可以揭示在体外切除DNA所需的最低酶组分。虽然以前的检测都是用纯化的加工酶成功切除的，但只使用裸DNA作为底物。这一新开发的切除试验将用于检测核小体模板，它更能反映体内基因组DNA的环境。这项研究旨在阐明DNA DSB修复的遗传和分子机制，特别是染色质和染色质重塑酶的作用。具体地说，它会问：核小体对DSB切除有什么影响，需要什么酶过程来克服这一障碍？通过确定克服染色质障碍所需的酶过程和招募顺序，将进一步加深对DSB修复的理解。在DSB修复过程中染色质和依赖于ATP的重构体的这种特征将允许通过靶向DSB的染色质重塑来促进同源重组来促进修复，从而加速开发用于癌症治疗的药物。 与公共健康相关：这项建议描述了一项研究，重点是染色体结构如何影响DNA断裂的修复，以及正常的细胞机制如何影响这种结构，影响DNA修复。具体地说，我们建议研究DNA切除过程中的染色质，这是同源重组所必需的。通过同源重组修复DNA是维持基因组完整性的关键途径之一，它已经被证明影响了一些人类的病理疾病，如年龄相关性疾病和某些类型的癌症。