Crystal structure of DNA-PKcs reveals a large open-ring cradle comprised of HEAT repeats

Abstract

Several members of the phosphatidylinositol-3-OH kinase (PI(3)K) family are involved in the response to DNA double-strand breaks. One of these, DNA-dependent protein kinase (DNA-PK), is comprised of three subunits, with the kinase activity residing in the catalytic subunit, DNA-PKcs. In this study, Tom Blundell and colleagues have solved the structure of human DNA-PKcs, at a resolution sufficient to see the overall folds. The structure reveals that the many HEAT repeats bend the protein into a circular structure. The kinase domain, encoded in the C-terminal domain, sits on one side of the structure. While the overall architecture of the catalytic subunit allows speculation about regions where conformational changes may occur, confirmation of such interactions awaits higher resolution data. If broken chromosomes arising from DNA double-strand breaks are left unrepaired or incorrectly repaired, they can lead to genomic changes that may result in cell death or cancer. DNA-dependent protein kinase (DNA-PK), which comprises the DNA-PK catalytic subunit (DNA-PKcs) and the heterodimer Ku70/Ku80, has a major role in the repair of double-strand breaks. The crystal structure of human DNA-PKcs is now presented, in which the overall fold is clearly visible. Broken chromosomes arising from DNA double-strand breaks result from endogenous events such as the production of reactive oxygen species during cellular metabolism, as well as from exogenous sources such as ionizing radiation1,2,3. Left unrepaired or incorrectly repaired they can lead to genomic changes that may result in cell death or cancer. DNA-dependent protein kinase (DNA-PK), a holoenzyme that comprises the DNA-PK catalytic subunit (DNA-PKcs)4,5 and the heterodimer Ku70/Ku80, has a major role in non-homologous end joining—the main pathway in mammals used to repair double-strand breaks6,7,8. DNA-PKcs is a serine/threonine protein kinase comprising a single polypeptide chain of 4,128 amino acids and belonging to the phosphatidylinositol-3-OH kinase (PI(3)K)-related protein family9. DNA-PKcs is involved in the sensing and transmission of DNA damage signals to proteins such as p53, setting off events that lead to cell cycle arrest10,11. It phosphorylates a wide range of substrates in vitro, including Ku70/Ku80, which is translocated along DNA12. Here we present the crystal structure of human DNA-PKcs at 6.6 Å resolution, in which the overall fold is clearly visible, to our knowledge, for the first time. The many α-helical HEAT repeats (helix–turn–helix motifs) facilitate bending and allow the polypeptide chain to fold into a hollow circular structure. The carboxy-terminal kinase domain is located on top of this structure, and a small HEAT repeat domain that probably binds DNA is inside. The structure provides a flexible cradle to promote DNA double-strand-break repair.