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

描述（由申请人提供）：在所有对基因组的损害中，dsb被认为是对细胞最有害的一种。它们既来自电离辐射或化疗药物等环境因素，也来自DNA复制、V(D)J重组或减数分裂等正常细胞过程。如果未修复或修复不当，dsb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，会显著增加患癌症的风险。在临床上，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有两种修复途径：非同源末端连接（NHEJ）和同源依赖修复（HDR）。正确选择修复途径对基因组的稳定性至关重要。这两种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需要广泛的加工来形成3' s尾。包括我的实验室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，许多重要的问题仍然知之甚少。首先，HDR和NHEJ在细胞周期的S-G2期都有活性，但尚不清楚是什么因素首先决定了DSB是被切除（对于HDR）还是被切除。其次，电离辐射和许多抗癌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因子稳定束缚，但修复不能完成或严重延迟。目前尚不清楚它们是否被困住了，或者是否可以被重新输送到切除HDR的地方。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。在此应用程序中，提出了三个具体目标

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.