Cryo-EM visualization of DNA-PKcs structural intermediates in NHEJ

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions.
• The nonhomologous end joining (NHEJ) pathway repairs DSBs through a series of regulated protein complex transitions.
• Core NHEJ factors initially form a long-range (LR) synaptic complex that transitions to a short-range state for DNA end alignment.

Purpose of the Study:

• To visualize and characterize key transition states of NHEJ complexes.
• To elucidate the role and conformational dynamics of DNA-PKcs during NHEJ.
• To reveal the regulatory cycle of DNA-PKcs in DSB repair.

Main Methods:

• Single-particle cryo-electron microscopy (cryo-EM).
• Structural analysis of multiple NHEJ complexes.
• Investigation of DNA-PKcs conformations and autophosphorylation effects.

Main Results:

• Three additional NHEJ transition states were visualized by cryo-EM.
• DNA-PKcs adopts distinct dimeric conformations throughout the NHEJ process.
• Autophosphorylation of DNA-PKcs induces conformational changes in the LR complex, promoting DNA break exposure and dissociation.

Conclusions:

• DNA-PKcs undergoes significant conformational changes during NHEJ.
• These dynamic changes are crucial for regulating DNA break exposure and processing.
• A comprehensive model for the DNA-PKcs regulatory cycle in NHEJ is proposed.