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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
1R01CA166677-01
Application #
8275973
Study Section
Special Emphasis Panel (ZRG1-CE-M (09))
Program Officer
Pelroy, Richard
Project Start
2012-04-01
Project End
2017-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
1
Fiscal Year
2012
Total Cost
$329,493
Indirect Cost
$121,993
Name
University of Texas Sw Medical Center Dallas
Department
Radiation-Diagnostic/Oncology
Type
Schools of Medicine
DUNS #
800771545
City
Dallas
State
TX
Country
United States
Zip Code
75390
Zhang, S; Matsunaga, S; Lin, Y-F et al. (2016) Spontaneous tumor development in bone marrow-rescued DNA-PKcs(3A/3A) mice due to dysfunction of telomere leading strand deprotection. Oncogene 35:3909-18
Yu, Lan; Shang, Zeng-Fu; Abdisalaam, Salim et al. (2016) Tumor suppressor protein DAB2IP participates in chromosomal stability maintenance through activating spindle assembly checkpoint and stabilizing kinetochore-microtubule attachments. Nucleic Acids Res 44:8842-8854
Yu, Lan; Shang, Zeng-Fu; Hsu, Feng-Ming et al. (2015) NSCLC cells demonstrate differential mode of cell death in response to the combined treatment of radiation and a DNA-PKcs inhibitor. Oncotarget 6:3848-60
Sui, Jiangdong; Lin, Yu-Fen; Xu, Kangling et al. (2015) DNA-PKcs phosphorylates hnRNP-A1 to facilitate the RPA-to-POT1 switch and telomere capping after replication. Nucleic Acids Res 43:5971-83
Lee, Kyung-Jong; Shang, Zeng-Fu; Lin, Yu-Fen et al. (2015) The Catalytic Subunit of DNA-Dependent Protein Kinase Coordinates with Polo-Like Kinase 1 to Facilitate Mitotic Entry. Neoplasia 17:329-38
Bunch, Heeyoun; Lawney, Brian P; Lin, Yu-Fen et al. (2015) Transcriptional elongation requires DNA break-induced signalling. Nat Commun 6:10191
Lin, Yu-Fen; Shih, Hung-Ying; Shang, Zengfu et al. (2014) DNA-PKcs is required to maintain stability of Chk1 and Claspin for optimal replication stress response. Nucleic Acids Res 42:4463-73
Lin, Yu-Fen; Nagasawa, Hatsumi; Little, John B et al. (2014) Differential radiosensitivity phenotypes of DNA-PKcs mutations affecting NHEJ and HRR systems following irradiation with gamma-rays or very low fluences of alpha particles. PLoS One 9:e93579
Sui, Jiangdong; Li, Mengxia; Qian, Chengyuan et al. (2014) Functional analysis of tanshinone IIA that blocks the redox function of human apurinic/apyrimidinic endonuclease 1/redox factor-1. Drug Des Devel Ther 8:2147-60
Li, Mengxia; Lin, Yu-Fen; Palchik, Guillermo A et al. (2014) The catalytic subunit of DNA-dependent protein kinase is required for cellular resistance to oxidative stress independent of DNA double-strand break repair. Free Radic Biol Med 76:278-85

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