Pathway Choice of DNA Double-Strand Break Repair

DNA双链断裂修复的途径选择

• 批准号: 8305249
• 负责人: DAVID J CHEN
• 金额: $ 32.95万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-09 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8305249
• 关键词: Am 80; Apoptosis; Attenuated; Binding; Cancer Biology; Cell Cycle; Cell Cycle Stage; Cell Death; Cells; Chromatin; Chromosome abnormality; Clinical; Complex; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA-PKcs; Data; Dissociation; Double Strand Break Repair; Environment; Enzymes; Eukaryotic Cell; Event; Excision; Frequencies; G2 Phase; G22P1 gene; Genes; Genomic Instability; Goals; Hereditary Disease; Homologous Gene; Human; Human Genetics; Human Genome; In Vitro; Incidence; Ionizing radiation; Ku Protein; Laboratories; Lead; Ligation; Malignant Neoplasms; Mediating; Metabolism; Modality; Modeling; Molecular; Mus; Mutation; Mycobacterium tuberculosis; Nonhomologous DNA End Joining; Pathway interactions; Pharmaceutical Preparations; Phase; Phosphorylation; Phosphorylation Site; Play; Process; Protein Kinase; Proteins; Radiation therapy; Recruitment Activity; Regulation; Role; S Phase; Site; Synapses; Testing; Therapeutic; Therapeutic Agents; Translating; XRCC2 gene; cancer cell; cancer therapy; carcinogenesis; helicase; homologous recombination; in vivo; insight; mutant; neoplastic cell; novel; prevent; recombinational repair; repaired; senescence; tumorigenesis

DESCRIPTION (provided by applicant): DNA double strand breaks (DSBs) are caused by endogenous (byproducts of cellular metabolism and replication associated errors) and exogenous (ionizing radiation and chemotherapeutic drugs) agents. Unrepaired or misrepaired DSBs can result in senescence, inducted apoptosis, or chromosomal aberrations, including translocations and deletions. These chromosomal aberrations can lead to genomic instability and tumorigenesis. To counteract the effects of DNA DSBs, two highly efficient DSB repair pathways have evolved in eukaryotic cells: non-homologous end-joining (NHEJ) and homologous recombination (HR). The NHEJ pathway utilizes several enzymes, including Ku70/80 and DNA-PKcs, that capture both DNA ends, bring them together in a synaptic complex, and facilitate direct ligation of the DSB. HR is initiated once 5'-3' resection of the DSB occurs. The 5'-3' resection creates ssDNA ends which are subsequently used for strand invasion and exchange into a homologous DNA template and once resolved, the DSB is fully repaired. One of the major unresolved questions in the field of DNA repair is the mechanism that modulates the pathway choice between NHEJ and HR. The goal of this proposal is to test our hypothesis that the binding of Ku70/80 to DSBs plays a key role in protecting DNA ends regardless of the cell cycle stage and that dissociation of Ku from DSBs is one of the mechanisms responsible for modulating pathway choice between NHEJ and HR. In G1, we hypothesize that Ku70/80 mediates NHEJ-mediated DSB repair, but in S/G2 phases of the cell cycle it protects DNA from non-specific end processing until it is actively displaced from DSB ends to allow DNA end resection and HRmediated DSB repair. To test this hypothesis, we propose the following specific aims: 1) To test the hypothesis that Ku70/80 is required for DNA end stability in S phase of the cell cycle. Furthermore, we will determine if Ku70/80 blocks DNA end processing by assessing its ability to block DNA end resection via the known human factors responsible for this process using model DNA substrates, including a mononucleosome chromatin substrate in vitro. 2) To further support our hypothesis that Ku70/80 must be displaced from DSB ends for DNA end resection and HR to initiate, we will test if these processes are attenuated if DSB ends are persistently occupied by Ku in vivo. 3) To determine the mechanism that modulates Ku70/80's dissociation from DSBs to allow the initiation of DNA end resection and HR in S/G2 phases of the cell cycle. Basic mechanistic insights into DSB repair mechanisms and the regulation of pathway choice for the repair of DSBs is important as DSB repair is paramount for protecting the human genome. This is supported by the observations that an increase in cancer frequency is observed in mice and humans with mutations in genes that encode proteins responsible for the repair of DSBs. Furthermore, induction of DSBs is used as a therapeutic modality for cancer treatment. Taken together, these underlie the importance of understanding the coordination and function of DSB repair and insights into repair mechanisms will ultimately translate into clinical targets and benefits. PUBLIC HEALTH RELEVANCE: DSBs are caused by endogenous and exogenous agents and the inability to repair DSBs can result in cell death, genomic instability, and carcinogenesis. Because of these deleterious events, cells have developed two major pathways to repair DSBs, termed non-homologous end-joining (NHEJ) and homologous recombination (HR).The importance of DSB repair proteins for protecting DNA is supported by the observations that a number of human genetic diseases and an increase in cancer incidence are associated with impaired DSB repair including mutations in factors that are specifically required for NHEJ and HR. Furthermore, induction of DSBs is used as a therapeutic modality for cancer treatment, as radiation therapy and many chemo-therapeutic agents produce DSBs to induce tumor cell death. The goal of this study is to understand one of the major unresolved questions in the field of DNA repair: what molecular events underlie the choice between HR and NHEJ pathways for repair of DSBs? The increased level of DNA damage in cancer cells and the use of DNA damaging agents in cancer treatment underlie the importance of DSB repair in cancer biology. Basic mechanistic insights into DSB repair mechanisms and the regulation of pathway choice for the repair of DSBs are likely to translate into new opportunities for cancer treatment.

描述（由申请人提供）：DNA双链断裂（DSB）是由内源性（细胞代谢的副产品和相关误差的副产物）和外源性（电离辐射和化学治疗药物）引起的。未修复或虚假的DSB可能导致衰老，诱导的凋亡或染色体畸变，包括易位和缺失。这些染色体畸变会导致基因组不稳定性和肿瘤发生。为了抵消DNA DSB的效果，在真核细胞中已经发展了两个高效的DSB修复途径：非同源末端连接（NHEJ）和同源重组（HR）。 NHEJ途径利用了包括Ku70/80和DNA-PKC在内的几种酶，可捕获两个DNA末端，将它们放在突触复合物中，并促进DSB的直接连接。 DSB的5'-3'切除次数启动HR 发生。 5'-3'切除会产生ssDNA末端，随后用于链入侵并交换为同源的DNA模板，一旦解决，DSB就会充分修复。 DNA修复领域中主要未解决的问题之一是调节NHEJ和HR之间的途径选择的机制。该提案的目的是检验我们的假设，即KU70/80与DSB的结合在保护DNA末端的关键作用无论细胞周期阶段如何，并且KU与DSB的解离是负责调节途径选择​​的机制之一。在NHEJ和HR之间。在G1中，我们假设KU70/80介导了NHEJ介导的DSB修复，但是在细胞周期的S/G2阶段中，它可以保护DNA免受非特异性端处理，直到它从DSB中积极地置于DSB末端以允许DNA末端切除并HRDDNA端次切除并HRDIDECTIED介导DSB维修。为了检验这一假设，我们提出了以下特定目的：1）检验以下假设：在细胞周期的S相中，DNA终端稳定性需要KU70/80。此外，我们将通过评估其通过使用模型DNA底物（包括单核小体染色质底物在体外）来阻止DNA终端切除的能力来确定KU70/80块DNA末端处理。 2）为了进一步支持我们的假设，即必须从DSB末端流离该DSB，以进行DNA终端切除和HR以启动，我们将测试如果ku in vivo持续占据DSB端，是否会减弱这些过程。 3）确定调节Ku70/80与DSB解离的机制，以允许在细胞周期的S/G2阶段开始DNA终端切除和HR。对DSB修复机制的基本机械洞察力和DSB修复途径选择的调节很重要，因为DSB修复对于保护人类基因组至关重要。这是由观察结果支持的，即在小鼠和人类中观察到癌症频率的增加，并在编码负责修复DSB的蛋白质的基因中观察到了突变。此外，DSB的诱导被用作癌症治疗的治疗方式。综上所述，这些是了解DSB修复的协调和功能以及对修复机制的洞察力的重要性，最终将转化为临床目标和益处。 公共卫生相关性：DSB是由内源性和外源性药物引起的，无法修复DSB会导致细胞死亡，基因组不稳定性和癌变。由于这些有害事件，细胞已经开发了两种主要途径来修复DSB，称为非同源末端连接（NHEJ）和同源重组（HR）。DSB修复蛋白在保护DNA中的重要性是由观察结果支持的，该数字是一个数字，该数字是一个数字人类遗传疾病和癌症发病率的增加与DSB修复受损有关，包括NHEJ和HR特别需要的因素的突变。此外，DSB的诱导被用作癌症治疗的治疗方式，作为放射治疗和许多化学治疗剂产生DSB，以诱导肿瘤细胞死亡。这项研究的目的是了解DNA修复领域的主要未解决问题之一：哪些分子事件是HR和NHEJ途径之间选择DSB之间的选择？癌细胞中DNA损伤水平的增加以及在癌症治疗中使用DNA损伤剂的使用是DSB修复在癌症生物学中的重要性。对DSB修复机制的基本机械洞察力和DSB修复途径选择的调节可能会转化为癌症治疗的新机会。