Cryo-EM Imaging of DNA-PK DNA Damage Repair Complexes

Abstract

Exposure to low levels of ionizing radiation causes DNA double-strand breaks (DSBs) that must be repaired for cell survival. Higher eukaryotes respond to DSBs by arresting the cell cycle, presumably to repair the DNA lesions before cell division. In mammalian cells, the nonhomologous end-joining DSB repair pathway is mediated by the 470 kDa DNA-dependent protein kinase catalytic subunit (DNA-PKcs) together with the DNA-binding factors Ku70 and Ku80. Mouse knock-out models of these three proteins are all exquisitely sensitive to low doses of ionizing radiation. In the presence of DNA ends, Ku binds to the DNA and then recruits DNA-PKcs. After formation of the complex, the kinase activity associated with DNA-PKcs becomes activated. This kinase activity has been shown to be essential for repairing DNA DSBs in vivo since expression of a kinase-dead form of DNA-PKcs in a mammalian cell line that lacks DNA-PKcs fails to complement the radiosensitive phenotype. The immense size of DNA-PKcs suggests that it may also serve as a docking site for other DNA repair proteins. Since the assembly of the DNA-PK complex onto DNA is a prerequisite for DSB repair, it is critical to obtain structural information on the complex. Cryo-electron microscopy (cryo-EM) and single particle reconstruction methods provide a powerful way to image large macromolecular assemblies at near atomic (10-15 ?) resolution. We have already used cryo-EM methods to examine the structure of the isolated DNA-PKcs protein. This structure reveals numerous cavities throughout the protein that may allow passage of single or double-stranded DNA. Pseudo two-fold symmetry was found for the monomeric protein, suggesting that DNA-PKcs may interact with two DNA ends or two Ku heterodimers simultaneously. Here we propose to study the structure of the cross-linked DNA-PKcs/Ku/DNA complex. Difference imaging with our published DNA-PKcs structure will enable us to elucidate the architecture of the complex. A second objective is to locate the kinase domain of DNA-PKcs by determining the structure of a kinase deletion mutant both as an isolated protein and as part of a DNA-PKcs/Ku/DNA complex. A third objective is to pursue higher resolution studies of DNA-PKcs and the DNA-PKcs/Ku/DNA complex. If the crystal structure determination of DNA-PKcs is completed during the project period, the atomic coordinates of DNA-PKcs will be modeled within the cryo-EM structure of the complex. In order to achieve these goals, a collaborative effort is proposed between Dr. Phoebe Stewart at UCLA, whose laboratory has expertise in cryo-EM reconstruction methods, and Dr. David Chen at the Lawrence Berkeley National Laboratory, who has a long-standing interest in DNA repair. Advantages of the cryo-EM structural method include the fact that the sample is imaged in a frozen-hydrated and unstained state, avoiding artifacts associated with drying and staining in other EM approaches. Also crystals of the sample are not needed for the single particle reconstruction method and only microgram quantities of sample are required. Cryo-EM structural information of macromolecular assemblies is complementary to both atomic structures of individual component molecules, as well as low resolution information obtained from x-ray and neutron scattering. Knowledge of the geometrical arrangement of the complex, and the position of the essential DNA-PKcs kinase domain, should lead to a greater understanding of the molecular events in DNA double-strand break repair following exposure to low doses of radiation.