DNA-PKcs Phosphorylation on Hematopoietic Stem Cells Genome Maintenance

描述(申请人提供)：DNA依赖蛋白激酶催化亚单位(DNA-PKcs)及其DNA结合伙伴Ku70/80异源二聚体是非同源末端连接(NHEJ)途径的关键组成部分。作为对DNA双链断裂(DSB)的响应，DNA-PKcs在T2609簇处迅速磷酸化，这是DSB修复的关键事件。DNA-PKcs“3A”敲击小鼠，其中小鼠T2605簇(人T2609簇)的三个残基的磷酸化通过丙氨酸替代被去除，由于先天性骨髓衰竭而过早死亡。DNA-PKcs3A/3A小鼠的造血干细胞(HSCs)丧失是由基因毒性应激增加引起的，表现为肠隐窝细胞凋亡增加和皮肤色素沉着。尽管这些小鼠过早死亡，但它们可以通过骨髓移植获救。骨髓移植挽救的DNA-PKcs3A/3A小鼠容易患血液性和非血液性癌症。HSC缺失和皮肤色素沉着是人类先天性角化不良综合征和小鼠端粒1b(POT1b)和端粒酶RNA(MTR)基因双重敲除的主要特征。此外，DC患者也容易发生癌症。DC患者和POT1b/MTR DKO小鼠无法正确维护端粒。基于这些发现，我们假设DNA-PKcs3A蛋白的表达将导致端粒失调、基因组不稳定和致癌。在这个项目中，我们建议进一步阐明DNA-PKcs T2609簇磷酸化影响HSC稳态和端粒维持的机制。我们的具体目标是：1.研究DNA-PKcs T2609簇的磷酸化以及DNA-PKcs与Ku70/80异二聚体的相互作用对造血干细胞动态平衡的影响。2.探讨DNA-PKcs T2609簇磷酸化对端粒维持的影响。3.检测DNA-PKcs T2609簇磷酸化对端粒3‘端突起的产生和保护的影响。

DESCRIPTION (provided by applicant): The DNA dependent protein kinase catalytic subunit (DNA-PKcs) and its DNA-binding partner the Ku70/80 heterodimer are the key components of the non-homologous end-joining (NHEJ) pathway. In response to DNA double-strand breaks (DSBs), DNA-PKcs is rapidly phosphorylated at the T2609 cluster, an event critical for DSB repair. DNA-PKcs "3A" knockin mice, in which phosphorylation at three residues in the mouse T2605 cluster (human T2609 cluster) are ablated via alanine substitution, die prematurely due to congenital bone marrow failure. Loss of hematopoietic stem cells (HSCs) in DNA-PKcs3A/3A mice is caused by elevated genotoxic stress as evidenced by increased intestinal crypt apoptosis and skin hyperpigmentation. Although these mice die prematurely, they can be rescued with bone marrow transplantation. BMT-rescued DNA- PKcs3A/3A mice are prone to develop both hematologic and non-hematologic cancers. HSC loss and hyperpigmented skin are the main features found in human dyskeratosis congenita (DC) syndrome and in mice double knockout of protection of telomeres 1b (POT1b) and telomerase RNA (mTR) genes. In addition, DC patients are also prone to cancer development. DC patients and POT1b/mTR DKO mice are unable to properly maintain the telomeres. Based on these findings, we hypothesize that the expression of the DNA-PKcs3A protein will lead to telomere dysregulation, genome instability, and carcinogenesis. We propose in this project to further elucidate the mechanism by which DNA-PKcs T2609 cluster phosphorylation impacts HSC homeostasis and telomere maintenance. Our specific aims are: 1. To investigate how DNA-PKcs T2609 cluster phosphorylation and the DNA-PKcs interaction with the Ku70/80 heterodimer impacts hematopoietic stem cell homeostasis. 2. To investigate the effect of DNA-PKcs T2609 cluster phosphorylation on telomere maintenance. 3. To determine the effect of DNA-PKcs T2609 cluster phosphorylation on the production and protection of telomeric 3' overhangs.