Homeodomain protein PRRX1 anchors the Ku heterodimers at DNA double-strand breaks to promote nonhomologous end-joining.

The DNA-dependent protein kinase (DNA-PK) complex plays a critical role in nonhomologous end-joining (NHEJ), a template-independent pathway for repairing DNA double-strand breaks (DSBs). The association of Ku70/80 with DSB ends facilitates the assembly of the DNA-PK holoenzyme. However, key mechanisms underlying the attachment and stabilization of DNA-PK at broken DNA ends remain unclear. Here, we identify PRRX1, a homeodomain-containing protein, as a mediator of chromatin localization and subsequent activation of DNA-PK. PRRX1 oligomerizes to simultaneously bind to double-strand DNA and the SAP (SAF-A/B, Acinus, and PIAS) domain of Ku70, thereby enhancing Ku anchoring at DSBs and stabilizing DNA-PK for efficient NHEJ repair. Reduced expression or pathogenic mutations of PRRX1 are associated with genomic instability and impaired NHEJ repair. Furthermore, a peptide that disrupts PRRX1 oligomerization compromises NHEJ efficiency and reduces cell survival following irradiation. These findings provide new insights into the activation of the NHEJ machinery and offer potential strategies for optimizing cancer therapies.