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

• 批准号: 8516047
• 负责人: Hong Yan
• 金额: $ 42.22万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8516047
• 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 Fingerprinting; 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; Genome; Genome Stability; Genomic Instability; 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; public health relevance; reconstitution; repaired; research study

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(申请人提供)：在所有对基因组的损害中，双链球菌被认为是对细胞最有害的之一。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。如果不修复或修复不当，双链断裂会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，BRCA1和BRCA2，极大地增加了癌症的风险。临床上，通过电离辐射和各种抗癌药物诱导DSB也是治疗癌症最常用的方法之一。DSB的修复一般有两种途径：非同源末端连接(NHEJ)和同源依赖修复(HDR)。修复途径的正确选择对基因组的稳定性至关重要。这两条通路分叉的关键事件是DNA末端的初始处理。NHEJ涉及有限的处理，但HDR需要广泛的处理才能形成3‘s尾。最近几个实验室的研究，包括我的，已经阐明了切除的基本机制。然而，许多重要的问题仍然鲜为人知。首先，HDR和NHEJ在细胞周期的S-G2期都是活跃的，但尚不清楚是什么因素首先决定了DSB是被引导到切除(对于HDR)还是被引导到NHEJ。其次，电离辐射和许多抗癌药物产生的末端经常携带受损的核苷酸。这些末端仍然可以被NHEJ因素稳定地结合，但修复不能完成或严重延迟。目前还不清楚他们是否被困住了，或者是否可以重新引导到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。特殊目的I旨在检验这样一种假设，即决定是否切除DSB的关键因素是末端的结构。末端与蛋白质加合物相连的DNA经常被许多抗癌药物诱导，将被用作检验这一假说的模型底物。它的修复将通过对非洲爪哇卵子提取物和纯化的切除蛋白进行生化重建研究来严格分析。特殊目的II旨在检验这样的假设，即以受损核苷酸结束时与NHEJ因子结合，然后通过Mre11-Rad50-NBS1(MRN)复合体重新引导到切除以进行HDR。被MRN介导的机制从末端移位的靶蛋白将被识别。《特定目的III》旨在验证沃纳综合征蛋白(WRN)和DNA2核酸酶的假说，这两个蛋白质和DNA2核酸酶对非洲爪哇卵提取物中的切除至关重要，对人类细胞的切除也是重要的。这些研究将大大增加对DNA末端如何被切除以及DSB修复途径是如何选择的理解。参与DSB切除和末端重导的关键蛋白可能被开发成新的药物靶点或作为生物标志物来提高肿瘤的放射治疗和化疗的效率。