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DNA Double-Strand Break Repair Pathway Choice and the Resection of DNA Ends

DNA 双链断裂修复途径选择和 DNA 末端切除

基本信息

批准号：
8854092
负责人：
Hong Yan
金额：
$ 43.76万
依托单位：
RESEARCH INST OF FOX CHASE CAN CTR
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-08-01 至 2017-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8854092
关键词：
Address Affinity Antineoplastic Agents BRCA1 gene BRCA2 gene Binding Binding Proteins Biochemical Biological Markers Biological Models Bloom Syndrome Bloom syndrome protein Cell Cycle Cell Death Cell physiology Cells Chemotherapy-Oncologic Procedure Chromosomal translocation Chromosome Deletion Complement Complex DNA DNA Double Strand Break DNA biosynthesis DNA-Binding Proteins Defect Development Double Strand Break Repair Drug Design Drug Targeting EXO1 gene Effectiveness Eukaryota Event Excision G2 Phase Gene Targeting Genes Genetic Fingerprintings Genome Genome Stability Genomic Instability Health Human Ionizing radiation Lead Left Link Maintenance Malignant Neoplasms Mediating Meiosis Methods Mining Mitochondria Modeling Mutation NBS1 gene Nonhomologous DNA End Joining Nuclear Nucleotides Oncogenic Pathway interactions Pharmaceutical Preparations Play Premature aging syndrome Process Proteins Public Health Radiation therapy Reaction Research Resected Role Series Structure System Tail Technology Testing V(D)J Recombination Werner Syndrome Xenopus adduct cancer radiation therapy cancer risk cancer therapy design ds-DNA egg environmental agent exodeoxyribonuclease helicase human WRN protein improved knock-down novel nuclease premature reconstitution repaired research study

项目摘要

DESCRIPTION (provided by applicant): Among all the damages to the genome, DSBs are considered one of the most deleterious to cells. They arise from both environmental agents like ionizing radiation or chemotherapeutic drugs and normal cellular processes like DNA replication, V(D)J recombination, or meiosis. If un-repaired or improperly repaired, DSBs would cause chromosome deletions or translocations, ultimately leading to premature cell death or oncogenic transformation. Mutations in many DSB repair genes, such as Werner syndrome gene and Bloom syndrome gene, BRCA1, and BRCA2, dramatically increase the risk of cancer. Clinically, eliciting DSBs by ionizing radiation and various cancer drugs is also among the most commonly used methods to treat cancer. DSBs are repaired by two general types of pathways: non-homologous end joining (NHEJ) and homology-dependent repair (HDR). Proper choice of repair pathway is critical to genome stability. The key event in the bifurcation of the two pathways is the initial processing of DNA ends. NHEJ involves limited processing, but HDR requires extensive processing to form 3' ss-tails. Recent studies from several labs, including mine, have elucidated the basic mechanisms for resection. However, many important questions are still poorly understood. Firstly, HDR and NHEJ are both active during S-G2 phases of the cell cycle, but it is unclear what factors first determine if a DSB is channeled to resection (for HDR) or to NHEJ. Secondly, ends generated by ionizing radiation and many cancer drugs often carry damaged nucleotides. These ends can still be stably bound by NHEJ factors, but repair cannot be completed or is seriously delayed. It is unclear if they are trapped or can be re-channeled to resection for HDR. Thirdly, compared to model systems, the understanding of resection in human cells is still very limited. In this application, three specific aims are proposed to address these important questions. Specific Aim I is designed to test the hypothesis that a key factor for determining if a DSB is resected or not is the structure of ends. DNA with ends linked to a protein adduct, which are frequently induced by many cancer drugs, will be used as a model substrate to test this hypothesis. Its repair will be rigorously analyzed by biochemical reconstitution studies in Xenopus egg extracts and with purified resection proteins. Specific Aim II is designed to test the hypothesis that ends with damaged nucleotides are bound by NHEJ factors but then re- channeled to resection for HDR by the MRE11-RAD50-NBS1 (MRN) complex. The target protein that is dislodged from ends by the MRN-mediated mechanism will be identified. Specific Aim III is designed to test the hypothesis that the Werner syndrome protein (WRN) and the DNA2 nuclease, which are critical for resection in Xenopus egg extracts, are also important for resection in human cells. These studies will greatly increase the understanding of how DNA ends are resected and consequently how DSB repair pathways are chosen. The key proteins involved in DSB resection and end re-channeling might be developed into new targets for drugs or as biomarkers to increase the efficiency of radiation therapy and chemotherapy of cancer.

描述（由申请人提供）：在对基因组的所有损害中，DSB被认为是对细胞最有害的损害之一。它们既来自环境因素，如电离辐射或化疗药物，也来自正常的细胞过程，如DNA复制、V（D）J重组或减数分裂。如果未修复或修复不当，DSB将导致染色体缺失或易位，最终导致细胞过早死亡或致癌转化。许多DSB修复基因的突变，如Werner综合征基因和Bloom综合征基因，BRCA 1和BRCA 2，显着增加癌症的风险。在临床上，通过电离辐射和各种癌症药物引发DSB也是治疗癌症最常用的方法之一。DSB通过两种一般类型的途径进行修复：非同源末端连接（NHEJ）和同源依赖性修复（HDR）。正确选择修复途径对基因组稳定性至关重要。两条途径分叉的关键事件是DNA末端的初始加工。NHEJ涉及有限的加工，但HDR需要大量加工以形成3'ss-尾。包括我在内的几个实验室最近的研究已经阐明了切除的基本机制。然而，许多重要问题仍然知之甚少。首先，HDR和NHEJ在细胞周期的S-G2期期间都是活跃的，但不清楚是什么因素首先决定DSB被引导到切除（对于HDR）还是NHEJ。其次，由电离辐射和许多癌症药物产生的末端通常携带受损的核苷酸。这些末端仍然可以被NHEJ因子稳定地结合，但修复不能完成或严重延迟。目前尚不清楚它们是否被困或是否可以重新引导以进行HDR切除。第三，与模型系统相比，对人体细胞切除的理解仍然非常有限。在本申请中，提出了三个具体目标来解决这些重要的问题。具体目标I旨在检验以下假设：决定是否切除DSB的关键因素是末端结构。DNA的末端连接到蛋白质加合物，这是经常诱导的许多癌症药物，将被用作模型基板来测试这一假设。它的修复将通过爪蟾卵提取物和纯化的切除蛋白的生化重建研究进行严格的分析。特异性目的II旨在检验以下假设：具有受损核苷酸的末端被NHEJ因子结合，但随后通过MRE 11-RAD 50-NBS 1（MRN）复合物重新引导至HDR切除。将鉴定通过MRN介导的机制从末端移位的靶蛋白。特定目的III旨在检验以下假设：沃纳综合征蛋白（WRN）和DNA 2核酸酶对非洲爪蟾卵提取物中的切除至关重要，对人类细胞中的切除也很重要。这些研究将大大增加对DNA末端如何被切除以及DSB修复途径如何选择的理解。参与DSB切除和末端再通道的关键蛋白可能被开发为新的药物靶点或生物标志物，以提高癌症的放射治疗和化疗的效率。