The DNA dependent protein kinase (DNA-PK) is central in non-homologous end joining (NHEJ), one of two major double strand break repair (DSBR) pathways in eukaryotes. DNA-PK recognizes double strand breaks (DSBs) and targets other factors to the site of damage. DNA-PK's enzymatic activity is critical to its function in NHEJ;however, to date, the catalytic subunit of DNA-PK (DNA-PKcs) itself is the only NHEJ factor known to be a functionally relevant target of its own enzymatic activity. Autophosphorylation of DNA-PKcs occurs on many sites, and is functionally complex;distinct phosphorylation events have distinct functional consequences. Phosphorylation within two major clusters allows the kinase to regulate (both positively and negatively) access of the damaged DNA to other NHEJ factors as well as to other DNA repair pathways, especially the other major DSBR pathway, homologous recombination (HR). Additional DNA-PK phosphorylation events are required for subsequent steps in NHEJ including kinase dissociation and inactivation. The first two aims of the proposed research will explore 1) how DNA-PK regulates DNA end access and 2) define and characterize other DNA-PK phosphorylation events that are required during DNA repair.
The third aim will focus on how DNA-PK regulates DSBR pathway choice, and characterize the biologic consequence of this choice in living animals. For one type of double strand break, there is no choice;DSBs introduced during VDJ recombination by the RAG endonuclease are exclusively repaired by NHEJ. Preliminary data suggest that the RAG complex targets its DSBs to NHEJ by interaction with DNA-PK.
The fourth aim will characterize how RAG induced breaks are restricted to the NHEJ pathway.There is an emerging consensus that the DNA dependent protein kinase (DNA-PK) is more stringently required in some species than in others, and it seems likely that DNA-PK deficiency is not compatible with human life. Understanding how this large complex functions to coordinate DNA repair by the non-homologous end joining pathway and potentially regulate access of DNA lesions to other repair pathways should provide fundamental knowledge of how DNA-PK promotes genomic stability.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI048758-13
Application #
8225205
Study Section
Special Emphasis Panel (ZRG1-IMM-J (02))
Program Officer
Nasseri, M Faraz
Project Start
2000-03-15
Project End
2013-05-14
Budget Start
2012-03-01
Budget End
2013-05-14
Support Year
13
Fiscal Year
2012
Total Cost
$372,438
Indirect Cost
$127,413
Name
Michigan State University
Department
Pathology
Type
Schools of Veterinary Medicine
DUNS #
193247145
City
East Lansing
State
MI
Country
United States
Zip Code
48824
Neal, Jessica A; Xu, Yao; Abe, Masumi et al. (2016) Restoration of ATM Expression in DNA-PKcs-Deficient Cells Inhibits Signal End Joining. J Immunol 196:3032-42
Meek, Katheryn; Xu, Yao; Bailie, Caleb et al. (2016) The ATM Kinase Restrains Joining of Both VDJ Signal and Coding Ends. J Immunol 197:3165-3174
Masani, Shahnaz; Han, Li; Meek, Katheryn et al. (2016) Redundant function of DNA ligase 1 and 3 in alternative end-joining during immunoglobulin class switch recombination. Proc Natl Acad Sci U S A 113:1261-6
Ying, Songmin; Chen, Zhihui; Medhurst, Annette L et al. (2016) DNA-PKcs and PARP1 Bind to Unresected Stalled DNA Replication Forks Where They Recruit XRCC1 to Mediate Repair. Cancer Res 76:1078-88
Roy, Sunetra; de Melo, Abinadabe J; Xu, Yao et al. (2015) XRCC4/XLF Interaction Is Variably Required for DNA Repair and Is Not Required for Ligase IV Stimulation. Mol Cell Biol 35:3017-28
Douglas, Pauline; Ye, Ruiqiong; Trinkle-Mulcahy, Laura et al. (2014) Polo-like kinase 1 (PLK1) and protein phosphatase 6 (PP6) regulate DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation in mitosis. Biosci Rep 34:
Neal, Jessica A; Sugiman-Marangos, Seiji; VanderVere-Carozza, Pamela et al. (2014) Unraveling the complexities of DNA-dependent protein kinase autophosphorylation. Mol Cell Biol 34:2162-75
Mahaney, Brandi L; Hammel, Michal; Meek, Katheryn et al. (2013) XRCC4 and XLF form long helical protein filaments suitable for DNA end protection and alignment to facilitate DNA double strand break repair. Biochem Cell Biol 91:31-41
Müller, Tina A; Yu, Kefei; Hausinger, Robert P et al. (2013) ALKBH1 is dispensable for abasic site cleavage during base excision repair and class switch recombination. PLoS One 8:e67403
Woodbine, Lisa; Neal, Jessica A; Sasi, Nanda-Kumar et al. (2013) PRKDC mutations in a SCID patient with profound neurological abnormalities. J Clin Invest 123:2969-80

Showing the most recent 10 out of 21 publications