Real-time visualization of G2L4 reverse transcriptase in DNA repair via microhomology-mediated end joining

The error-prone microhomology-mediated end joining (MMEJ) pathway for double-strand break repair (DSBR) is important for genome integrity in all organisms. The bacterial group II intron-like reverse transcriptase, G2L4 RT, has been implicated in MMEJ, but how it executes DSBR is not fully known. Using high-speed atomic force microscopy (HS-AFM), we directly visualize G2L4 RT-mediated DSBR via MMEJ. We observe that G2L4 RT dimers exhibit apparent RT3a plug protrusion from the active site upon DNA engagement and catalyze MMEJ by binding and stabilizing short microhomologies and filling adjacent single-strand gaps with dNTPs. Furthermore we find a Mn²⁺-stimulated terminal transferase activity that generates elongated and branched DNA intermediates prior to ligation. Using T4 DNA ligase, we visualize ligase binding near nick sites and real-time nick sealing, which stabilizes the repaired products and suppresses off-pathway branching. These results reveal how G2L4 RT and DNA ligase activities shape MMEJ intermediates and outcomes.