DNA-PKcs Phosphorylation on Hematopoietic Stem Cells Genome Maintenance

描述（由申请人提供）：DNA依赖性蛋白激酶催化亚基（DNA-PKcs）及其DNA结合配偶体Ku 70/80异二聚体是非同源末端连接（NHEJ）途径的关键组分。响应于DNA双链断裂（DSB），DNA-PKcs在T2609簇处迅速磷酸化，这是DSB修复的关键事件。DNA-PKcs“3A”敲入小鼠，其中小鼠T2605簇（人T2609簇）中三个残基的磷酸化通过丙氨酸取代被消除，由于先天性骨髓衰竭而过早死亡。DNA-PKcs 3A/3A小鼠中造血干细胞（HSC）的丢失是由升高的遗传毒性应激引起的，如肠隐窝凋亡增加和皮肤色素沉着过度所证明的。虽然这些小鼠过早死亡，但可以通过骨髓移植来挽救它们。BMT拯救的DNA-PKcs 3A/3A小鼠易于发展血液学和非血液学癌症。HSC丢失和皮肤色素沉着是人类先天性角化不良（DC）综合征和端粒1b（POT 1b）和端粒酶RNA（mTR）基因保护双敲除小鼠中发现的主要特征。此外，DC患者也容易发生癌症。DC患者和POT 1b/mTR DKO小鼠无法正确维持端粒。基于这些发现，我们推测DNA-PKcs 3A蛋白的表达将导致端粒失调、基因组不稳定和致癌。本研究旨在进一步阐明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簇磷酸化及与Ku 70/80异源二聚体的相互作用对造血干细胞稳态的影响。2.探讨DNA-PKcs T2609簇磷酸化对端粒维持的影响。3.确定DNA-PKcs T2609簇磷酸化对端粒3'突出端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.