Pathway Choice of DNA Double-Strand Break Repair

描述(申请人提供)：DNA双链断裂(DSB)是由内源性(细胞代谢和复制相关错误的副产品)和外源性(电离辐射和化疗药物)因素引起的。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异常，包括易位和缺失。这些染色体异常可导致基因组不稳定和肿瘤的发生。为了抵消DNADSB的影响，在真核细胞中进化出两条高效的DSB修复途径：非同源末端连接(NHEJ)和同源重组(HR)。NHEJ途径利用几种酶，包括Ku70/80和DNA-PKcs，它们捕获DNA的两端，将它们聚集在一个突触复合体中，并促进DSB的直接连接。一旦DSB切除5‘-3’，就开始HR 发生。5‘-3’切除产生单链DNA末端，这些末端随后用于链入侵并交换到同源DNA模板中，一旦溶解，DSB就被完全修复。DNA修复领域中尚未解决的主要问题之一是调控NHEJ和HR之间的途径选择的机制。这个建议的目的是验证我们的假设，即Ku70/80与DSB的结合在保护DNA末端方面发挥着关键作用，而不受细胞周期阶段的影响，并且Ku从DSB中解离是调节NHEJ和HR之间路径选择的机制之一。在G1期，我们假设Ku70/80介导了NHEJ介导的DSB修复，但在细胞周期的S/G2期，Ku70/80保护DNA免受非特异性末端加工，直到它从DSB末端主动移位，从而允许DNA端切除和HR介导的DSB修复。为了检验这一假说，我们提出了以下具体目标：1)检验细胞周期S期DNA末端稳定性所必需的Ku70/80的假说。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，通过评估Ku70/80阻止DNA末端切除的已知人类因素的能力，来确定Ku70/80是否阻止DNA末端处理。2)为了进一步支持我们的假设，即DNA末端切除和HR必须从DSB末端移位Ku70/80，我们将在体内测试如果DSB末端持续被Ku占据，这些过程是否被减弱。3)探讨细胞周期中KU70/80‘S从DSB解离，从而启动细胞周期中DNA末端切除和细胞周期中HR的机制。由于DSB修复对保护人类基因组至关重要，因此对DSB修复机制和DSB修复途径选择的调节是重要的。观察到，在编码负责DSB修复的蛋白质的基因突变的小鼠和人类中，癌症频率增加，这支持了这一点。此外，DSB的诱导被用作癌症治疗的一种治疗方式。综上所述，这些都强调了了解DSB修复的协调和功能的重要性，对修复机制的洞察最终将转化为临床目标和好处。

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.