Autophosphorylation transforms DNA-PK from protecting to processing DNA ends.

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• DNA-dependent protein kinase (DNA-PK) plays a crucial role in DNA double-strand break repair via non-homologous end joining (NHEJ).
• Processing of DNA hairpin ends during V(D)J recombination requires the Artemis nuclease and DNA-PK activity.

Purpose of the Study:

• To elucidate the structural and functional mechanisms by which DNA-PK regulates DNA end processing during NHEJ.
• To understand how DNA-PK interacts with Artemis and DNA structures to control V(D)J recombination.

Main Methods:

• X-ray crystallography was used to determine the structures of DNA-PK in various states: bound to open DNA ends, autophosphorylated, and in complex with Artemis and a DNA hairpin.
• Biochemical assays were employed to assess DNA-PK kinase activity and phosphorylation status.

Main Results:

• DNA-PK exhibits distinct conformational states and activities depending on the DNA end structure it binds.
• Binding to open DNA ends inhibits DNA-PK cis-autophosphorylation but activates other phosphorylation events.
• DNA hairpin ends promote DNA-PK cis-autophosphorylation, leading to structural rearrangements that facilitate Artemis recruitment and hairpin opening.
• Artemis binding to phosphorylated DNA-PK locks it in a kinase-inactive state, coordinating repair progression.

Conclusions:

• DNA-PK undergoes a functional switch regulated by DNA end structure and phosphorylation status.
• This switch mechanism allows DNA-PK to coordinate DNA end protection and processing, ensuring efficient and accurate NHEJ and V(D)J recombination.