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

描述（由申请人提供）：染色质和atp依赖性重塑在DNA双链断裂加工中的作用DNA双链断裂（DSBs）的修复被认为是维持基因组完整性的关键过程。dsb可以由环境因素（电离辐射、诱变化学物质等）和/或内源性因素（氧自由基、DNA复制分叉断裂或减数分裂等程序化细胞过程）引起。这种修复途径的缺陷会导致基因易位和染色体重排等突变，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，先前的研究表明，染色质重塑复合体Ino80的募集是DSB正常修复所必需的。Ino80的募集依赖于组蛋白H2AX的磷酸化，并被认为通过可能改变断裂附近染色质的结构来促进DSB的修复。我们的初步结果表明，Ino80的招募本身可能依赖于DSB加工。本项目将研究Ino80在单个特定位点上被诱导到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Ino80招募的要求。为了确定染色质对DSB修复的影响，将采用纯化DSB处理酶的体外系统。这种最近开发的生化分析允许揭示体外切除DNA所需的最低酶成分。虽然以前的分析成功切除纯化的加工酶，只有裸DNA被用作底物。这种新开发的切除试验将用于检查核小体模板，更能反映基因组DNA在体内的环境。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：核小体对DSB切除的影响是什么？需要什么样的酶促过程来克服这一屏障？通过确定克服染色质屏障所需的酶促过程和招募顺序，将进一步了解DSB修复。对DSB修复过程中染色质和ATP依赖性重塑因子的描述，将有助于开发治疗癌症的药物，通过靶向DSB的染色质重塑，通过同源重组促进修复。

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.