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

描述（由申请人提供）：染色质和ATP依赖性重塑对DNA双链断裂加工的作用已知DNA双链断裂（DSB）的修复是维持基因组完整性的关键过程。DSB可由环境因素（电离辐射、诱变化学品等）和/或内源性来源（氧自由基、塌陷的DNA复制叉或编程的细胞过程如减数分裂）。这种修复途径的缺陷可能导致突变，如基因易位和染色体重排，从而导致癌症。虽然最近的研究揭示了DSB修复的一些过程，但确切的机制尚未阐明。此外，尽管表征了参与DSB修复的加工酶，但染色质在该过程中的作用目前尚不明确。一种流行的理论认为，组蛋白和/或核小体的丢失是DSB加工所必需的。为了支持这一理论，以前的研究表明，招募Ino 80，染色质重塑复合物，是正确的DSB修复所必需的。Ino 80募集依赖于组蛋白H2 AX磷酸化，并且被认为通过可能改变邻近断裂的染色质结构来促进DSB修复。我们的初步结果表明，Ino 80招聘本身可能依赖于DSB处理。本项目将研究Ino 80在单个特定位点募集到诱导DSB的遗传要求。该分析将在存在和不存在关键加工酶的情况下进行，以更清楚地描述Ino 80募集的要求。为了确定染色质对DSB修复的影响，将采用使用纯化的DSB加工酶的体外系统。这种最近开发的生物化学测定允许在体外切除DNA所需的最小酶组分的揭示。虽然先前的测定用纯化的加工酶实现了成功的切除，但仅使用裸DNA作为底物。这种新开发的切除试验将用于检查核小体模板，这更能反映体内基因组DNA的环境。本研究旨在阐明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。具体来说，它会问：什么是DSB切除的核小体的效果，需要什么酶的过程来克服这一障碍？通过确定酶的过程和招聘需要克服染色质的障碍，了解DSB修复将进一步。DSB修复期间染色质和ATP依赖性重塑物的这种表征将允许通过可能靶向DSB处的染色质重塑以促进通过同源重组的修复来加速开发用于治疗癌症的药理学试剂。 公共卫生相关性：该提案描述了研究重点是染色体结构如何影响DNA断裂的修复，以及正常细胞机制如何影响这种结构，从而影响DNA修复。具体而言，我们提出了在DNA切除，这是同源重组所需的染色质的研究。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，其已显示影响几种病理性人类病症，如年龄相关疾病和某些类型的癌症。

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.