Real-Time Visualization of G2L4 Reverse Transcriptase in DNA Repair via Microhomology-Mediated End Joining

Double-strand break repair (DSBR) is essential for genome integrity, yet mechanistic details of error-prone microhomology-mediated end joining (MMEJ) remain unclear. A bacterial group II intron-like reverse transcriptase, G2L4 RT, has been implicated in MMEJ, but how it executes DSBR is unknown. Using high-speed atomic force microscopy (HS-AFM), we directly visualize G2L4 RT-mediated DSBR via MMEJ. We observe that G2L4 RT dimers exhibit RT3a plug protrusion upon DNA engagement and catalyze MMEJ by binding and stabilizing a 4-bp annealed microhomology and filling adjacent single-strand gaps with dNTPs. We also observe Mn ²⁺ -stimulated terminal transferase activity that generates elongated and branched DNA intermediates prior to ligation. With T4 DNA ligase, we visualize 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 ligase activities shape MMEJ intermediates and outcomes.