Chromosome end protection by RAP1-mediated inhibition of DNA-PK

Area of Science:

• Molecular biology
• Genetics
• Cell biology

Background:

• Classical non-homologous end joining (cNHEJ) repairs DNA double-strand breaks using DNA-dependent protein kinase (DNA-PK) and ligase 4 (LIG4).
• DNA-PK interacts with telomeres but does not initiate cNHEJ, suggesting context-specific regulation.
• The precise mechanisms controlling DNA-PK activity at telomeres remain elusive.

Purpose of the Study:

• To elucidate the molecular mechanism regulating DNA-PK function at telomeres.
• To identify factors that prevent telomere end joining and maintain chromosome integrity.

Main Methods:

• Biochemical assays to study protein-DNA interactions.
• Cryo-electron microscopy to determine complex structures.
• Cell-based assays in mouse and human cells to assess cNHEJ activity.

Main Results:

• TRF2 and RAP1 form a complex with DNA-PK, directly inhibiting its end-joining function.
• RAP1, when bound to TRF2, prevents DNA-PK from recruiting LIG4 through interactions with KU and DNA.
• RAP1 acts redundantly with the Apollo nuclease in repressing cNHEJ at chromosome ends.

Conclusions:

• DNA-PK's end-joining activity is specifically repressed at telomeres by the TRF2-RAP1 complex.
• This repression occurs independently of overhang-dependent mechanisms, providing a parallel pathway for telomere protection.
• A molecular mechanism is established for maintaining individual linear chromosomes in mammalian cells by regulating DNA-PK at telomeres.