Pathway Choice of DNA Double-Strand Break Repair

描述（由申请人提供）：DNA双链断裂（DSB）由内源性（细胞代谢和复制相关错误的副产物）和外源性（电离辐射和化疗药物）试剂引起。未修复或错误修复的DSB可导致衰老、诱导的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双链断裂的影响，在真核细胞中已经进化出两种高效的双链断裂修复途径：非同源末端连接（NHEJ）和同源重组（HR）。NHEJ途径利用几种酶，包括Ku 70/80和DNA-PKcs，其捕获两个DNA末端，将它们一起形成突触复合物，并促进DSB的直接连接。一旦切除DSB的5 '-3'，就启动HR 发生。5 '-3'切除产生ssDNA末端，其随后用于链侵入和交换成同源DNA模板，并且一旦解析，DSB被完全修复。在DNA修复领域中的一个主要未解决的问题是调节NHEJ和HR之间的途径选择的机制。80至DSB在保护DNA末端方面起关键作用，而不管细胞周期阶段如何，并且Ku从DSB的解离是负责调节NHEJ和HR之间的途径选择的机制之一。在G1期，我们假设Ku 70/80介导NHEJ介导的DSB修复，但在细胞周期的S/G2期，它保护DNA免受非特异性末端加工，直到它从DSB末端被主动置换，以允许DNA末端切除和HR介导的DSB修复。为了验证这一假设，我们提出了以下具体目标：1）验证细胞周期S期DNA末端稳定性需要Ku 70/80的假设。此外，我们将确定是否Ku 70/80块DNA末端加工，通过评估其能力，通过已知的人为因素负责这个过程中使用模型DNA基板，包括单核细胞染色质基板在体外阻断DNA末端切除。2)为了进一步支持我们的假设，即Ku 70/80必须从DSB末端置换以进行DNA末端切除和HR启动，我们将测试如果DSB末端在体内被Ku持续占据，这些过程是否减弱。3)确定调节Ku 70/80从DSB解离以允许启动DNA末端切除和细胞周期S/G2期HR的机制。对DSB修复机制和DSB修复途径选择的调节的基本机械见解是重要的，因为DSB修复对于保护人类基因组至关重要。这得到了以下观察结果的支持：在编码负责DSB修复的蛋白质的基因突变的小鼠和人类中观察到癌症频率增加。此外，DSB的诱导被用作癌症治疗的治疗方式。总之，这些都是理解DSB修复的协调和功能的重要性的基础，对修复机制的了解最终将转化为临床目标和益处。

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.