Nonhomologous end joining (NHEJ) serves as the primary pathway for repairing DNA double-strand breaks (DSBs) in humans. Repairing DNA damage that occurs from oxidative damage and exposure to ionizing radiation is vital for genetic stability and for suppression of oncogenesis. NHEJ is also essential for V-D-J recombination in lymphocytes, which generates a functional adaptive immune system. The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) regulates repair the NHEJ pathway along with other key components Ku and Artemis. The Ku70/80 heterodimer is the first protein to recognize and bind DNA ends at double strand breaks and recruits DNA-PKcs to the damage sites. Artemis in complex with DNA-PKcs performs the endonucleolytic activity necessary for the hairpin-opening step of V-D-J recombination and DNA end processing in NHEJ. Mutations in any of these three components results in radiosensitivity and severe combined immunodeficiency in humans. The lack of high resolution structural information on DNA- PKcs and NHEJ complexes has prevented a mechanistic understanding of their critical DNA repair activity and regulation. CryoEM single particle image reconstruction is well suited for studying DNA-PKcs and large NHEJ complexes.
The specific aims of this proposal are to determine subnanometer (<10E) resolution cryoEM structures of DNA-PKcs/Artemis/DNA, DNA-PKcs/Artemis, DNA-PKcs/dsDNA, and DNA- PKcs/Ku/DNA complexes, as well as perform an atomic level structural analysis of these NHEJ complexes with emerging tools from the protein structure prediction field. The structural analysis will include docking of available atomic resolution structures and comparative models, as well as application of hybrid cryoEM de novo protein structure prediction methods. These studies will be highly complementary to ongoing biochemical, genetics, and x-ray crystallographic studies. Detailed knowledge of the molecular geometry of these complexes will provide insight into the kinase activation and endonuclease phases of NHEJ and will enable generation of testable hypotheses on molecular mechanisms underlying DNA break repair by the NHEJ pathway. Ultimately our ability to therapeutically treat cancer and immunodeficiency diseases will be enhanced by a molecular understanding of the underlying biological processes that are improperly regulated in the disease state.

Public Health Relevance

The proposed studies are biomedically relevant in that structural information on NHEJ complexes will help to answer key questions on how these complexes assemble at DNA damage sites, how the repair and recombination processes are guided, and what triggers the choice between multiple parallel pathways and outcomes. Ultimately this information will be helpful in understanding and treating cancer, severe combined immune deficiency (SCID), and sensitivity to ionizing radiation (RS-SCID).

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
7R01CA140538-03
Application #
8225317
Study Section
Radiation Therapeutics and Biology Study Section (RTB)
Program Officer
Knowlton, John R
Project Start
2010-05-04
Project End
2014-08-31
Budget Start
2012-03-01
Budget End
2013-08-31
Support Year
3
Fiscal Year
2012
Total Cost
$231,862
Indirect Cost
$84,179
Name
Case Western Reserve University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Lindert, Steffen; Stewart, Phoebe L; Meiler, Jens (2013) Computational determination of the orientation of a heat repeat-like domain of DNA-PKcs. Comput Biol Chem 42:1-4
Woetzel, Nils; Lindert, Steffen; Stewart, Phoebe L et al. (2011) BCL::EM-Fit: rigid body fitting of atomic structures into density maps using geometric hashing and real space refinement. J Struct Biol 175:264-76