Pathway Choice of DNA Double-Strand Break Repair

DNA双链断裂修复的途径选择

基本信息

批准号：
8457051
负责人：
DAVID J CHEN
金额：
$ 31.01万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
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/8457051
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

项目摘要

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.

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复制相关错误）和外源性（电离辐射和化疗药物）试剂引起的。未修复或错误修复的 DSB 可导致衰老、诱导细胞凋亡或染色体畸变，包括易位和缺失。这些染色体畸变可导致基因组不稳定和肿瘤发生。为了抵消 DNA DSB 的影响，真核细胞中进化出了两种高效的 DSB 修复途径：非同源末端连接 (NHEJ) 和同源重组 (HR)。 NHEJ 途径利用多种酶，包括 Ku70/80 和 DNA-PKcs，它们捕获两个 DNA 末端，将它们组合在一起形成突触复合体，并促进 DSB 的直接连接。 DSB 5'-3' 切除后开始 HR 发生。 5'-3'切除产生单链DNA末端，随后用于链侵入并交换成同源DNA模板，一旦解析，DSB就被完全修复。 DNA 修复领域尚未解决的主要问题之一是调节 NHEJ 和 HR 之间路径选择的机制。该提案的目的是检验我们的假设，即无论细胞周期阶段如何，Ku70/80 与 DSB 的结合在保护 DNA 末端方面发挥着关键作用，并且 Ku 从 DSB 解离是负责调节途径选择的机制之一NHEJ 和 HR 之间。在 G1 中，我们假设 Ku70/80 介导 NHEJ 介导的 DSB 修复，但在细胞周期的 S/G2 阶段，它保护 DNA 免受非特异性末端加工，直到它从 DSB 末端主动移位，以允许 DNA 末端切除和 HR 介导的修复DSB修复。为了检验这一假设，我们提出以下具体目标：1) 检验细胞周期 S 期 DNA 末端稳定性需要 Ku70/80 的假设。此外，我们将使用模型 DNA 底物（包括体外单核小体染色质底物）评估 Ku70/80 阻止 DNA 末端切除的能力，从而确定 Ku70/80 是否通过负责该过程的已知人为因素来阻断 DNA 末端加工。 2）为了进一步支持我们的假设，即Ku70/80必须从DSB末端置换才能进行DNA末端切除和HR启动，我们将测试如果DSB末端在体内持续被Ku占据，这些过程是否会减弱。 3) 确定调节 Ku70/80 从 DSB 解离的机制，以允许在细胞周期的 S/G2 期启动 DNA 末端切除和 HR。 DSB 修复机制的基本机制和 DSB 修复途径选择的调节非常重要，因为 DSB 修复对于保护人类基因组至关重要。观察结果支持了这一点，即在编码负责 DSB 修复的蛋白质的基因发生突变的小鼠和人类中，癌症发生率增加。此外，DSB的诱导被用作癌症治疗的治疗方式。总而言之，这些奠定了理解 DSB 修复的协调和功能的重要性，并且对修复机制的深入了解最终将转化为临床目标和益处。