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

Abstract

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 holo-enzyme that comprises DNA-dependent protein kinase catalytic subunit (DNA-PKcs)4, 5 and the heterodimer Ku70/Ku80, plays a major role in non-homologous end joining (NHEJ), 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 4128 amino acids and belonging to the phosphotidyl inositol 3-kinase (PI3-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 6.6Å resolution, in which the overall fold is for the first time clearly visible. 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 C-terminal kinase domain is located on top of this structure and a small HEAT repeat domain that likely binds DNA is inside. The structure provides a flexible cradle to promote DNA double-strand-break repair.

Structural insights into nonsense-mediated mRNA decay (NMD) by electron microscopy

“…SMG1 and the SMG1-SMG9 complex appeared in EM as "question mark" shaped-molecules with a large "head" region and a protruding "arm" (Figure 2a). SMG1 C-terminal end was located at the "head" region, which showed similarities with the C-terminus of DNA-PKcs (DNA-dependent Protein Kinase catalytic subunit), a member of the PIKK family that has been extensively characterized [33]. SMG1 Nterminal end was arranged as an "arm" extending from the catalytic C-terminus.…”

Structural characterization of filaments formed by human Xrcc4–Cernunnos/XLF complex involved in nonhomologous DNA end-joining

“…The Ku70/Ku80 heterodimer adopts a preformed ring-shaped structure that recognizes and encircles the duplex DNA ends at the DSB (7). Ku70/Ku80 recruits a 469-kDa serine/threonine protein kinase, the DNA-PK catalytic subunit (DNA-PKcs), via a direct interaction and shifts about 10 bp inward so that DNA-PKcs acquires a position at the terminus through its large open-ring cradle structure (8). Upon association with Ku and DNA, DNA-PKcs is activated and phosphorylates several proteins including itself and Ku70/Ku80.…”

Reprint of “The clinical impact of deficiency in DNA non-homologous end-joining”

“…Ku-DNA binding is, therefore, DNA sequence independent. The structure of DNA-PKcs has revealed a flexible ring shaped molecule arising from multiple alpha-helical HEAT repeats (helix-turn-helix motifs), which facilitate bending [9]. The flexible structure and an opening in the ring endows DNA-PKcs with the capacity to fold onto the DNA molecule [10].…”