DNA-PKcs Phosphorylation on Hematopoietic Stem Cells Genome Maintenance

DNA-PKcs 磷酸化对造血干细胞基因组维护的影响

• 批准号: 8275973
• 负责人: Benjamin Ping-Chi Chen
• 金额: $ 32.95万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8275973
• 关键词: Ablation; Adult; Alanine; Apoptosis; Apoptotic; Attenuated; Binding; Biological Preservation; Bone Marrow Transplantation; Catalytic Domain; Cell Death; Cells; Complex; DNA; DNA Binding; DNA Double Strand Break; DNA Repair; DNA-PKcs; DNA-dependent protein kinase; Development; Disease; Double Strand Break Repair; Dyskeratosis Congenita; Embryo; Eukaryota; Event; Failure; Functional disorder; G22P1 gene; Genes; Genome; Genome Stability; Genomic Instability; Genotoxic Stress; Hematologic Neoplasms; Hematopoietic; Hematopoietic stem cells; Heterogeneous-Nuclear Ribonucleoproteins; Homeostasis; Human; Hyperpigmentation; Hypersensitivity; Invaded; Investigation; Ionizing radiation; Knock-out; Knockout Mice; Lead; Length; Life; Maintenance; Malignant Neoplasms; Mediating; Metabolism; Mus; Mutation; Non-Hematologic Malignancy; Nonhomologous DNA End Joining; Pancytopenia; Pathway interactions; Patients; Phosphorylation; Production; Proliferating; Proteins; Skin; Staining method; Stains; Stress; Stretching; Structure; Syndrome; Telomerase RNA Component; Telomere Length Maintenance; Telomere Maintenance; Work; XRCC5 gene; base; carcinogenesis; hnRNP A1; homologous recombination; intestinal crypt; mouse model; null mutation; response; telomere

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. PUBLIC HEALTH RELEVANCE: Telomere length preservation is essential to sustain the life of eukaryotes. Dysregulation of telomere often leads to cell death, genome instability, and carcinogenesis. Investigation how DNA-PKcs and its phosphorylation impacts telomere length maintenance will facilitate our understanding on the mechanism of telomere metabolism and the pathophysiology of bone marrow failure diseases.

描述（由申请人提供）：DNA依赖性蛋白激酶催化亚基（DNA-PKC）及其DNA结合伴侣KU70/80异二聚体是非同源端粘合（NHEJ）途径的关键组成部分。为了响应DNA双链断裂（DSB），DNA-PKC在T2609群集上迅速磷酸化，这是DSB修复至关重要的事件。 DNA-PKCS“ 3A”敲击蛋白小鼠，其中小鼠T2605簇（人T2609簇）在三个残基上的磷酸化是通过丙氨酸取代消化的，由于先天性骨髓衰竭而过早死亡。 DNA-PKCS3A/3A小鼠中造血干细胞（HSC）的丧失是由遗传毒性应激升高引起的。尽管这些小鼠过早死亡，但可以通过骨髓移植来救出。 BMT验证的DNA-PKCS3A/3A小鼠容易发展血液学和非血液学癌症。 HSC丧失和色素沉重的皮肤是人类心脏病病（DC）综合征的主要特征，而小鼠的端粒保护1B（POT1B）和端粒酶RNA（MTR）基因的保护双重敲除。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。根据这些发现，我们假设DNA-PKCS3A蛋白的表达将导致端粒失调，基因组不稳定性和癌变。我们在该项目中建议进一步阐明DNA-PKCS T2609簇磷酸化影响HSC稳态和端粒维持的机制。我们的具体目的是：1。研究DNA-PKCS T2609簇磷酸化和DNA-PKCS与KU70/80异二聚体的相互作用如何影响造血干细胞稳态。 2。研究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'突出的产生和保护的影响。 公共卫生相关性：端粒长度保存对于维持真核生物的寿命至关重要。端粒的失调通常会导致细胞死亡，基因组不稳定性和致癌作用。研究DNA-PKC及其磷酸化如何影响端粒长度维持将有助于我们对端粒代谢机制和骨髓衰竭疾病的病理生理学的理解。